PAPER 208 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: betalain, microbial population, quality, red beetroot, sanitation, total phenolic - PHYSICOCHEMICAL, MICROBIAL AND SENSORY QUALITY OF FRESH-CUT RED BEETROOTS IN RELATION TO SANITIZATION METHOD AND STORAGE DURATION DULAL CHANDRA*, AE JIN CHOI, YONG PHIL KIM and JI GANG KIM Postharvest Research Team, National Institute of Horticultural and Herbal Science, Rural Development Administration, 203 Cheoncheon-ro, Jangan-gu, Suwon 440-706, Republic of Korea *Corresponding author: dchandra@korea.kr; dchandrajp@gmail.com ABSTRACT Effects of sanitization and storage on fresh-cut beetroots (Beta vulgaris L.) were evaluated fol- lowing sanitation – peeling - cutting (SPC), peeling – sanitation – cutting (PSC) and peeling – cut- ting – sanitation (PCS) methods with (Cl), or without (TW), 100 ppm chlorine solution, then pack- aged in polyethylene bag and stored at 5°C for up to 14 days. Chroma values of fresh-cut beet- roots significantly declined whereas whiteness index and titratable acidity values increased, how- ever, texture and total soluble solid contents showed no significant variation. Betalain contents decreased gradually and total phenol content showed inconsistence trend. PCS-Cl treated sam- ples accounted for higher betalains decline and received lower visual quality scores despite its low- er total aerobic bacterial count. Minimum microbial population was observed in PSC-Cl methods along with higher levels of betalain contents. Considering pigment retention, microbial and visu- al qualities, beetroots sanitized with chlorine water following PSC method was the best process- ing way for fresh-cut beetroots and therefore, PSC-Cl treatment could commercially be used for processing of fresh-cut beetroots. mailto:dchandra@korea.kr mailto:dchandrajp@gmail.com Ital. J. Food Sci., vol. 27 - 2015 209 INTRODUCTION Beet is a root vegetable of the Chenopodiaceae family whose edible part is its tuberous root. Its purple-red color is due to the presence of beta- lain pigments. These pigments are similar to an- thocyanins and flavonoids, which were wrong- ly termed in the old literature as anthocyanins containing nitrogen. Betalains are water-solu- ble, vacuolar pigments and are found only in ten families of the Centrospermae group and are di- vided into two classes: red betacyanin and yel- low betaxanthin (FENENA, 1995). In addition to their color, betalains possess several desirable biological activities including antioxidant, anti- inflammatory, hepatoprotective, and antitumor properties (ESCRIBANO et al., 1998; WINKLER et al., 2005). Although some studies have indicat- ed their potential as antioxidant pigments, beta- lains have not been much explored because of their relative scarceness in nature. The bioavail- ability of betalains was reported to be high in hu- mans, and they remain stable in the gastrointes- tinal tract without any significant loss in antiox- idative properties, which increases their value as health food additives (FRANK et al., 2005; PAV- LOV et al., 2005). Among other compounds with antioxidant properties, phenolics are believed to act as antioxidant, anti-carcinogenic, anti-mi- crobial, anti-mutagenic and anti-inflammato- ry, as well as in the reduction of cardiovascu- lar diseases (YANG et al., 2001; KIM et al., 2003; VALI et al., 2007). Because of these nutritional importances of plant phenolics, there has been an increasing interest in the evaluation of their changes with postharvest treatment (CHAUDRY et al., 1998; LEWIS et al., 1999). However, liter- atures are scanty on phenolic content of plant foods, especially of roots vegetables, which are important constituents of diets in many coun- tries. Storage and processing can reduce the content of phenolic compounds as some of them are easily oxidized, while others are more sta- ble. Processing in the form of simple peeling of fruit and vegetables can remove a major portion of the phenols, as the concentrations of these substances are often higher in the outer than the inner parts (MANACH et al., 2004). Pigment distribution in beetroots, on the other hand, also varied substantially among the outer, middle and center tissues, the former contained much higher pigments than the later tissues (GAERT- NER and GOLDMAN, 2005). Beet was traditionally used as a vegetable boiled in stews, baked in tarts, roasted as a whole and cut into salads. This vegetable has recently been using as a minimally processed food in many countries. However, the main tech- nological problems of processing fresh-cut beet roots are the significant discoloration and de- hydration of the minimally processed material. Washing and rinsing operations carried out after slicing have favored the loss of betacyanin and betaxanthin, since these pigments are soluble in water (NILSON, 1970). Moreover, minimally pro- cessed produces are more perishable than their whole counterparts, due to undergoing severe physical stresses especially during peeling and cutting procedures. Minimal processing com- prises selection, washing, peeling and cutting procedures that are aimed at producing a fresh and convenient product to prepare and consume (BURNS, 1995). The quality of fresh-cut produce depends on many factors, such as the state of the original plant (variety, plant part and mat- uration stage), harvesting date and storage, en- vironmental factors and processing techniques (MOURE et al., 2001). However, rapid quality de- terioration and shorter shelf-life are major prob- lems facing the industry for maintaining the quality and safety of fresh-cut produces. Sever- al studies have shown that fresh-cut produces are particularly susceptible to microbial growth owing to the removal of plant protective tissues and the release of cellular fluids from cutting (CARLIN et al., 1989; HEARD, 2002). The cut slic- es of beetroot become vulnerable to microbial at- tack, moisture loss and dehydration because of their large surface area. To ensure the microbial safety, use of proper sanitizing agents along the processing line is an important step in fresh-cut produce processing. Chlorine has been used for sanitation purposes in food processing industry for several decades and perhaps the most wide- ly used sanitizer in food industry. It is inexpen- sive, convenient to use and works against many food borne pathogens. Liquid chlorine and hy- pochlorite generally used in the 50 to 200 mg L-1 concentration range with a contact time of 1 to 2 min. ADAMS et al. (1989) reported that wash- ing lettuce leaves with 100 mg L-1 free chlorine can reduce population of aerobic mesophiles by more than 98% as compared to 92% reduction in tap water without chlorine. The use of beetroots as a fresh-cut produce is relatively new, especially in Asian countries. The information on various quality parameters of fresh-cut beetroots processed with different sanitization methods is limited. Hence, the objec- tives of this study were to find out the best pro- cessing and sanitation way for maintaining some physicochemical, microbial and sensory quali- ties, especially pigment and phenolic contents of fresh-cut beetroots during storage at 5°C. MATERIALS AND METHODS Preparation of sample Beetroots (Beta vulgaris L. var. Udan) sam- ples were harvested from a commercial farm in Jeju island, Republic of Korea, and were trans- ported to our laboratory within two days. Sam- ples were thoroughly washed in running tap water and subjected to different ways of sani- 210 Ital. J. Food Sci., vol. 27 - 2015 tation treatments such as sanitation – peeling - cutting (SPC), peeling – sanitation – cutting (PSC) and peeling – cutting – sanitation (PCS) with (Cl), or without (TW), 100 ppm chlorinat- ed solution. Chlorinated solution was prepared with NaOCl (100 ppm free chlorine, pH 7.0) as a standard industrial disinfection treatment for fresh-cut produces. After peeling out, beetroot samples were cut into small pieces (ca. 4 × 5 × 1 cm) excluding the top and bottom most por- tions. Sanitation treatments lasted for 2 min. To remove the excess surface water, all sanitized samples were spread over a sieve like plastic tray (40 × 50 cm) previously washed and disinfected with the same sanitizing solution and allowed at room temperature. Fresh-cut beetroots sam- ple of about 300 g were packaged in 80 µm ny- lon polyethylene bag (25 × 20 cm) and thermal- ly sealed. Four replicates of each bag per treat- ment and storage duration (0, 3, 7, 10 and 14 days of storage) were prepared and stored in a dark cold room at 5°C. On each evaluation day, outer and inner tissues were separated from the fresh-cut red beetroots slices discarding the mid- dle portion and were stored at -80°C until need- ed for biochemical analyses. Color and texture measurement Using a chromameter (Minolta CR-400, Mi- nolta, Osaka, Japan), color readings were tak- en from the middle portion of both sides of sliced red beetroots on each evaluation day. Three pieces of sliced beetroot from each pouch were randomly selected and six readings (from both sides of each piece) were taken from each replicate and a total of 24 readings were av- eraged from each treatment on the measure- ment day. The chromameter was calibrated us- ing the manufacturer’s standard white plate (Y 93.5, x 0.3155, y 0.3320). Color changes were quantified in the L*, a*, b* color space. L* re- fers to the lightness and ranges from black = 0 to white = 100. A negative value of a* indicates green, while a positive number indicates red color. Positive and negative b* indicate yellow and blue color, respectively. The color values were further converted into Chroma value (OZ- TURK et al., 2009) and ‘Whiteness Index’ (WI) (BOLIN and HUXSOLL, 1991) and were com- puted by the formulae: Chroma = (a*2 + b*2)1/2; WI = 100 – [(100 - L*)2 + a*2 + b*2]1/2 The texture of fresh-cut red beetroot was measure in term of force required to make a puncture hole horizontally on the sliced beet- roots. The test was conducted through destruc- tive puncture test performance using a tex- ture analyzer (TA Plus, Lloyd Instruments, Am- etek Inc., UK) following the method of HAMP- SHIRE et al. (1987). Each sliced beetroots sample was placed horizontally on the stationary plat- form of the analyzer for the test. Tests were car- ried out on the middle part of each sliced beet- roots with a 4 mm diameter stainless steel cy- lindrical probe. The movement of the probe was adjusted to 5, 2 and 10 mm/s as the pre-test, test and post-test speed, respectively. With a running load cell of 100 N, the probe was at- tached to a creep meter equipped with the soft- ware (NEXYGEN™MT v 4.5, Lloyd Instruments, Ametek Inc., UK) for automatic analysis using a computer. The maximum amount of force (N) needed to make a puncture hole on the sliced beetroot was recorded. Five pieces of sliced beet- roots from each pouch were tested and a total of 20 data from four replicates were averaged from each treatment. All measurements were taken at room temperature (20°±2°C). Determination of total soluble solid, pH, and titratable acidity Total soluble solid (TSS), pH and titratable acidity (TA) were measured according to the AOAC (1980) procedures. On each evaluation day, about 5 pieces of sliced beetroot from each bag were cut into small pieces and wrapped with 2 layers of cotton cloth and placed in a Juice maker (Fru-X80, GooJung Chromatech Inc., Korea) attached to an air supplier (EvaA– 0300) of the same company. Then pressure was created by the air supplier to obtain a homog- enized solution of beetroots sample. TSS of the resultant cleared juice was measured in terms of °Brix using a refractometer (PAL–1, Atago Co. Ltd, Tokyo, Japan). The pH was determined us- ing a pH meter (D-55122, Schott Instruments GmbH, Germany) with a glass electrode. Titrat- able acidity was measured by potentiometric ti- tration with 0.1 N NaOH up to pH 8.2 using 10 mL juice and the results were expressed as per- centage of citric acid. Determination of betalains (betacyanin and betaxanthin) and total phenolic contents The methodology used for the determination of betacyanin and betaxanthin were adopted from VON ELBE (2001). Five gram of previously frozen samples from the outer and inner parts of sliced beetroots were macerated separately in 15 mL distilled water using an Ultra-Turrax tis- sue homogenizer (T 25 B, Ika Works Sdn. Bhd, Malaysia) at a moderate speed for about 1 min. The homogenate was transferred to a volumet- ric flask filtered through Whatman no. 1 filter paper placed on a glass funnel. The filter cake was washed several times with distilled water until the extract became colorless. The extract volume was adjusted with distilled water. The resulting beetroots juice or tissue extract was diluted with 0.05 M phosphate buffer, pH 6.5 such that the absorbance of beetroots juice at Ital. J. Food Sci., vol. 27 - 2015 211 538 nm was in between 0.4 and 0.5 AU. Final- ly, the absorbance of beetroots juice was meas- ured at 476, 538 and 600 nm with an UV-VIS recording spectrophotometer (DU 650, Beckman Coulter™, USA) and 0.05 M phosphate buffer, pH 6.5 was used as the blank. Values of beta- cyanin and betaxanthin amounts were obtained through the equation: x = 1.095 × (a – c), y = b – z – x/3.1, z = a – x where a = sample reading at 538 nm; b = sam- ple reading at 476 nm; c = sample reading at 600 nm; x = betacyanin absorption; y = betax- anthin absorption; z = impurities absorption (VON ELBE, 2001). Total phenolic compound was determined based on the method described by SINGLETON and ROSSI (1965) with few modifications. Five gram previously frozen sample from the outer and inner parts of fresh-cut red beetroots slic- es were separately homogenized with 80% eth- anol using an Ultra-Turrax tissue homogeniz- er (T 25 B, Ika Works Sdn. Bhd, Malaysia) at a moderate speed for about 1 min. The homoge- nate was incubated at 60°C water bath for 30 min and centrifuged at 15,000 × g for 15 min at 20°C and then the supernatant was collected in a volumetric flask. The homogenized tissue was re-extracted with 80% ethanol at the same way and the resulting supernatants were mixed to- gether and carefully made known volume with 80% ethanol. For the determination, Folin-Cio- calteu reagent was diluted with distilled water to make 1 N phenol reagent. In a test tube, 1 mL supernatant was diluted with 8 mL distilled water and 1 mL of 1 N phenol reagent was add- ed followed by mixing. After 5 min, 1 mL 15% sodium carbonate solution was added, mixed well and allowed the mixture at room tempera- ture (~20°C) for 2 h. The absorbance was read at 725 nm using an UV-VIS recording spectro- photometer (DU 650, Beckman Coulter™, USA). The concentration of total phenol was calculat- ed using standard curve of gallic acid and ex- pressed as gallic acid equivalents in mg 100 g-1 fresh weight. Monitoring of microbial population Microbiological counts were carried out on every sampling day including washing day. Fresh-cut beetroots pouches were aseptical- ly opened using a sterilized scissors dipped in 95% ethanol and then ignited in the flame of a Bunsen burner. Twenty grams beetroots sample was weighed out from each pouch and placed in a stomacher bag (Masher-bag P-LTS, BAC- cT ®, NBT, Japan). The beetroots sample was diluted 1:9 in double distilled autoclaved wa- ter and homogenized in a stomacher (Seward Stomacher 400C, Brinkmann, USA) for 1 min at 230 rpm. A 10-fold serial dilution was also made from the homogenate and 1 mL of ho- mogenate solution was inoculated onto total aerobic bacterial (TAB) count PetrifilmTM (3M Microbiology Products, St. Paul, Minn., USA). The plates were then incubated at 35°C for 48 h and the developing colonies (about 25 – 250) were counted with the assistance of a 3M micro- bial colony counter (same company as of plate) and reported as colony forming units (CFU) per gram of sample. Similarly, 1 mL homogenate was plated onto 3M PetrifilmTM yeast and mold (YM) count plates (same company) and incu- bated for 72 h at 25°C. After incubation, yeast and mold colonies were counted manually ac- cording to the instruction guide of the compa- ny. Colonization data were then converted to log CFU per gram of fresh sample. Sensory evaluation The sensory analysis of fresh-cut beetroots sample was carried out by an 8-member (aged 24 - 48) expert panel. The members of the pan- el were trained to recognize and score off-odor and overall visual quality of fresh-cut beetroots prior to the test. Off-odor was evaluated imme- diately after opening the packages and scored on a 5-point scale in which 0 = none, 1 = slight, 2 = moderate, 3 = strong, and 4 = extremely strong (LOPEZ-GALVEZ et al., 1997); a score of 3 was considered non-acceptable. Overall visual qual- ity was evaluated by using 9-point scale (9 = ex- cellent, 7 = good, 5 = fair, 3 = poor and 1 = unus- able) (GONZALEZ-AGUILAR et al., 1999). A score of 6 was considered as the limit of marketability. Statistical analysis The experiment was conducted with four replications per treatment. Statistical analy- ses of the data were carried out using SAS soft- ware (SAS Institute, Cary, NC, USA). The level of significance was calculated from the F value of ANOVA. Mean comparison was achieved by Duncan’s multiple range test. Prior to the final experiment, two preliminary experiments were conducted with limited replications that result- ed similar trend. RESULTS AND DISCUSSION Texture, total soluble solids, pH and titratable acidity The effects of different washing methods and storage duration on the textural proper- ties of fresh-cut red beetroots are presented in Table 1. The forces were almost similar for all the sanitization methods, except for an in- significant (P>0.05) lower value was record- ed in PSC-Cl treatment on washing day. How- ever, the values were slightly increased to an 212 Ital. J. Food Sci., vol. 27 - 2015 insignificant level at the end of 14 days stor- age. PCS-Cl treated sample exhibited the high- est value among the treatments at the end of storage. Maintaining texture of fresh-cut pro- duces is one the main criteria which is affect- ed by morphology, cell turgor, cells wall-mid- dle lamella structure, water content, biochem- ical components and also by the genetic back- ground of plant species (HARKER et al., 1997). Peeling and cutting of vegetable exposes the interior tissues and drastically increase the rate of evaporation of water. However, we ob- served less than 1% moisture loss of the orig- inal weight at the end of storage (data not shown). Apart from water loss, the slight in- crease in texture value during storage of fresh- cut beetroots in our study might be due to the lignification of cells that rendered harder tis- sues and as a result of wound healing at cut surfaces. Packaging fresh-cut produces with suitable polyethylene film and selecting ap- propriate storage temperature have shown to preserve quality. The insignificant increases in texture of fresh-cut beetroots indicate that our packaging film and storage temperature were appropriate. There was no significant difference (P>0.05) found in total soluble solid (TSS) content in fresh-cut beetroots regardless of sanitization treatment and storage duration, whereas slight variations were observed in pH and titratable acidity (TA) values among the treatments and Table 1 - Changes in some physicochemical qualities of fresh-cut redbeet root during storage at 5°C after processed with dif- ferent sanitization methods. Parameter/storage day Sanitization method SPC-TW SPC-Cl PSC-TW PSC-Cl PCS-TW PCS-Cl Texture (N) 0 52.58aA 52.22aA 49.92aA 47.61aA 51.13aA 52.49aA 7 52.83aA 53.80aA 51.40aA 49.51aA 52.47aA 53.85aA 14 53.43aA 54.97aA 53.39aA 50.36aA 54.29aA 55.36aA °Brix 0 8.50aA 8.67aA 7.83aA 8.70aA 8.33aA 8.23aA 7 8.23aA 8.60aA 8.37aA 8.03aA 8.57aA 8.53aA 14 7.90aA 8.67aA 8.03aA 8.27aA 8.43aA 8.37aA pH 0 6.28aA 6.25aAB 6.24bAB 6.28aA 6.22aAB 6.16cB 7 6.42aAB 6.38aBC 6.38aBC 6.30aC 6.35aBC 6.50aA 14 6.26aA 6.28aA 6.21bA 6.30aA 6.21aA 6.36bA TA (% citric acid) 0 0.10bA 0.10bA 0.10bA 0.12aA 0.09bA 0.09bA 7 0.09bAB 0.08bB 0.08bB 0.10aAB 0.11bA 0.10bAB 14 0.17aA 0.15aA 0.15aA 0.16aA 0.18aA 0.14aA SPC = sanitation - peeling - cutting, PSC = peeling - sanitation - cutting, PCS = peeling - cutting - sanitation, Cl = sanitation with 100 ppm chlorinated water, TW = sanitation with tap water. Values represent the mean of four replicates. Means under the same heading in each column or row with different small or capital letters, respectively are sig- nificantly different according to the Duncan test (P<0.05). different storage durations (Table 1). These reflect that although TSS was maintained throughout the storage, TA values increased significantly at the end of the storage that re- sulted smaller decline in pH values. The lowest TSS (7.83°Brix) was found in PSC-TW treated sample on washing day while the highest val- ue (8.70°Brix) was recorded in PSC-Cl treated sample on the same day. It was reported that TSS of fresh-cut beetroots are varied depend- ing on the cut type (KLUGE et al., 2006), sani- tization period (VITTI et al., 2011) and storage temperature (VITTI et al., 2005). Although our TSS data were little higher than that of VIT - TI et al. (2011) and VITTI et al. (2005) studies with fresh-cut beetroots, we found both simi- lar amount and changing pattern of TSS dur- ing storage as reported by KLUGE et al. (2006). The differences in TSS in this study and other studies might be due to the differences in soil condition, cultivar and sowing time (FELLER and FINK, 2004). The pH and TA values ranges from 6.16 to 6.50 and 0.08 to 0.18, respectively among the processing methods and through- out the storage (Table 1). In general, pH values slightly increased in the middle of storage and decreased later to their initial values. TA val- ues expressed as percentage of citric acid, on the other hand, remained unchanged until the middle of storage and then increased about 1.5 fold at the end of storage to their initial levels. Among the treatments, PCS-Cl treated sam- Ital. J. Food Sci., vol. 27 - 2015 213 ple showed the lowest values of pH and TA on washing day and 14 days of storage, respec- tively. In agreement with our results, LOPEZ OSORNIO and CHAVES (1998) found no varia- tion in pH values over a 7-day storage period of raw grated beetroots at 4°C and packaged in trays wrapped with polyvinylchloride film. However, they found steadily increased values of TA throughout the storage. Again, PILON et al. (2006) reported that the contents of ti- tratable acidity were not affected by the stor- age period in minimally processed carrot while the pH values decreased at the beginning of storage and increased thereafter to their ini- tial levels at the end of 3-week storage peri- od. The smaller range of changes in citric acid content during storage of fresh-cut beetroots might be accounted for lower respiratory ac- tivity, which is the indicator of retardation of overall metabolic activities. Color parameters Produce color is one of the most important quality factors that directly affect consum- ers’ choice. In this study, the effects of differ- ent sanitization methods on the color param- eters of fresh-cut beetroots were measured in term of chroma value as well as whiteness index and presented in Fig. 1. Among the treatments, SPC-Cl and PSC-TW showed significantly higher (P<0.05) chroma value on washing day. Howev- er, chroma values declined gradually in all treat- ments when storage progressed except few fluc- tuations on 7 and 14 days of storage. There was a significant (P<0.05) decline in chroma value in PCS-Cl treatment when storage progressed ex- hibiting the lowest values of chroma through- out the storage among the treatments. This re- sult implies that sanitation after cutting yielded higher loss of pigment in beetroots slices there- Fig. 1 - Changes in chroma value (above), whiteness index (below) of fresh-cut red beetroot after processing with different sanitation methods and during storage at 5°C. Legends: SPC = sanitation–peeling-cutting, PSC = peeling–sanitation–cutting, PCS = peeling–cutting–sanitation, Cl = sanitation with 100 ppm chlorinated water, TW = sanitation with tap water. 214 Ital. J. Food Sci., vol. 27 - 2015 by decreased the intensity of color. Between the other two sanitation treatments using chlorine water, PSC-Cl showed no significant decline in chroma value until the end of storage except on 10-day while SPC-Cl showed gradual decrease until 10-day followed by a slight increase at the end of storage (Fig. 1). The decrease in color is a consequence of loss of betalains, the main pig- ments of red beetroots (VON ELBE, 2001). There- fore, we found significant variation and decrease in betalain contents both on washing day and subsequent storage of fresh-cut beetroots in this study (Table 2 and later discussion). In consist- ence with our result, gradual decrease in color index of fresh-cut beetroots was reported previ- ously (VITTI et al., 2005; KLUGE et al., 2006; VIT- TI et al., 2011). Since chroma represents color saturation, which varies from dull (low value) to vivid color (high value), we used chroma val- ue as an indicator of freshness and purity of the color of beetroots slices. However, LOPEZ OS- ORNIO and CHAVES (1998) found significant in- crease in chroma values during storage of grat- ed beetroots. This might be attributed to the dif- ferences in processing, subsequent packaging and storage condition of beetroots in the present study with their study. Whiteness index (WI) on the other hand, sharply increased on the third day of storage in all samples (Fig. 1). Both of the sanitized and water washed samples of peeling – cutting - sanitation (PCS) method exhibited sig- nificantly (P<0.05) higher values of WI from the washing day to 10 days of storage. This result also indicates that higher pigment loss was oc- curred when beetroots samples were subject- ed to sanitize after cutting. Other two methods showed similar values of WI both on processing day and throughout the storage. At the end of the storage, WI values reached nearly similar level for all samples. Although WI was first measured for lignin formation on the surface of fresh-cut carrot slices (BOLIN and HUXSOLL, 1991), LOPEZ OSORNIO and CHAVES (1998) reported that the whitish substances in grated beetroots are a protective lignin biochemically synthesized af- ter peeling. It was also reported that WI is the most sensitive and easily measured indicator of sensory quality of fresh-cut carrot. Betalains (betacyanin and betaxanthin) content Significantly higher betalain contents were found in the outer tissues than in the inner tissues of fresh-cut beetroots slices (Table 2). Beetroots samples processed with peeling – cut- ting - sanitation (PCS) method exhibited signif- icantly (P<0.05) lower amount of betalain (be- tacyanin and betaxanthin) contents on wash- ing day compared to other methods. In general, sanitation – peeling – cutting (SPC) method en- sured higher amount of betalain contents than other methods whereas the betalain contents Ta b le 2 - C h a n ge s in b et a la in ( b et a cy a n in a n d b et a x a n th in ) co n te n t in t h e o u te r a n d i n n er t is su es o f fr es h -c u t re d b ee tr o o ts p ro ce ss ed w it h d if fe re n t sa n it iz a ti o n m et h o d s a n d d u r- in g st o ra ge a t 5 °C . Tr ea tm en t B et al ai n co nt en t/s to ra ge d ay B et ac ya ni n (m g 10 0g -1 F W ) B et ax an th in (m g 10 0g -1 F W ) To ta l b et al ai n (m g 10 0g -1 F W ) 0 3 7 10 14 0 3 7 10 14 0 3 7 10 14 O ut er ti ss ue S P C -T W 74 .6 5a A 65 .4 8a A B 55 .7 9a B C 54 .5 7a B C 49 .4 1a C 60 .5 4a A 54 .2 6a A 51 .6 4a B 47 .3 3a B C 42 .9 5a C 13 5. 19 aA 11 9. 74 aA B 10 7.4 3a B C 10 1. 90 aB C 92 .3 6a C S P C -C l 72 .8 5a bA 62 .0 8a A B 60 .3 7a A B 59 .7 0a A B 51 .9 1a B 56 .3 0a A 54 .5 3a A 51 .1 5a A B 49 .1 2a A B 44 .1 3a B 12 9. 15 ab A 11 6. 61 aA B 11 1. 52 aA B 1 08 .8 2a A B 96 .0 4a B P S C -T W 65 .3 5a bA 55 .8 0a A 53 .3 1a A 52 .4 9a A 49 .0 3a A 52 .5 0a A 51 .2 1a A 47 .3 5a A 41 .6 2a A 42 .4 9a A 11 7.8 5a bA 10 7.0 1a A 10 0. 66 aA 94 .1 1a A 91 .5 2a A P S C -C l 66 .7 2a bA 55 .5 0a A 53 .3 6a A 54 .9 6a A 54 .9 5a A 50 .9 1a A 45 .6 0a A 45 .4 9a A 43 .4 2a A 41 .8 8a A 11 7.6 3a bA 10 1. 10 aA 98 .8 5a A 98 .3 8a A 96 .8 3a A P C S -T W 58 .0 6b A 53 .7 5a A 52 .4 7a A 49 .1 3a A 47 .9 2a A 46 .4 6a A 41 .7 8a A 39 .7 8a A 44 .3 7a A 39 .7 4a A 10 4. 52 bA 95 .5 3a A 92 .2 5a A 93 .5 0a A 87 .6 6a A P C S -C l 57 .6 5b A 51 .5 2a A 48 .4 2a A 45 .0 2a A 42 .8 0a A 47 .8 7a A 43 .8 6a A 45 .1 7a A 40 .3 4a A 39 .2 5a A 10 5. 52 bA 95 .3 8a A 93 .5 9a A 85 .3 6a A 82 .0 5a A In ne r tis su e S P C -T W 37 .4 5a bA 34 .1 8a A B 34 .7 8a A B 30 .6 5a A B 25 .3 8a B 29 .8 2a bA 28 .3 1a A 28 .6 0a A 23 .7 4a A B 18 .7 2a B 67 .2 7a bA 62 .4 9a A 63 .3 8a A 54 .3 9a A B 44 .1 0a B S P C -C l 39 .8 8a A 31 .6 1a A B 32 .4 6a bA B 2 9. 43 aB 29 .2 4a B 32 .3 0a A 23 .9 1a B C 26 .8 8a A B 21 .4 1a B C 18 .0 8a C 72 .1 8a A 55 .5 2a B 59 .3 4a bA B 5 0. 84 aB 47 .3 2a B P S C -T W 30 .8 8b cA 30 .5 3a A 30 .8 9a bA 26 .0 3a A 25 .2 7a A 25 .3 1a bA 22 .8 0a A 24 .0 9a A 19 .3 4a A 18 .9 3a A 56 .1 9b cA 53 .3 3a A 54 .9 8a bA 45 .3 7a A 44 .2 0a A P S C -C l 28 .5 0c A 27 .7 1a A 31 .9 3a bA 26 .7 6a A 28 .1 5a A 25 .9 3a bA 21 .1 9a A B 21 .0 4a A B 20 .7 6a A B 18 .3 5a B 54 .4 3b cA 48 .9 0a A 52 .9 7a bA 47 .5 2a A 46 .5 0a A P C S -T W 28 .0 6c A 26 .8 8 aA 24 .0 3b A 24 .7 2a A 25 .2 9a A 22 .1 9b A 20 .3 3a A 21 .5 2a A 19 .6 8a A 17 .9 6a A 50 .2 5c A 47 .2 1a A 45 .5 5b A 44 .4 0a A 43 .2 5a A P C S -C l 25 .4 8c A 25 .7 9 aA 24 .3 9a bA 26 .1 8a A 23 .3 7a A 22 .1 7b A 22 .9 8a A 21 .7 5a A 19 .7 6a A 18 .4 5a A 47 .6 5c A 48 .7 7a A 46 .1 4a bA 45 .9 4a A 41 .8 2a A S P C = s an ita tio n – pe el in g - c ut tin g, P S C = p ee lin g – sa ni ta tio n – cu tti ng , P C S = p ee lin g – cu tti ng – s an ita tio n, C l = s an ita tio n w ith 1 00 p pm c hl or in at ed w at er , T W = s an ita tio n w ith ta p w at er . Va lu es re pr es en t t he m ea n of fo ur re pl ic at es . M ea ns u nd er th e sa m e he ad in g in e ac h co lu m n or ro w w ith d iff er en t s m al l o r c ap ita l l et te rs , r es pe ct iv el y ar e si gn ifi ca nt ly d iff er en t a cc or di ng to th e D un ca n te st (P <0 .0 5) . Ital. J. Food Sci., vol. 27 - 2015 215 of peeling – sanitation – cutting (PSC) meth- ods were in between the betalain contents lev- els of SPC and PCS methods. However, beta- lain contents gradually decreased when stor- age progressed in all washing/sanitation treat- ments. The rate of betalain decline during stor- age was higher in SPC sanitization method com- pared to other two methods thereby significant (P<0.05) declines were observed in all compo- nents of both tissues at the end of storage (Ta- ble 2). The highest (34.4%) decline of total beta- lain was found in both of SPC-TW and SPC-Cl treated samples of inner tissues while the low- est (12.2%) decline was observed in PCS-Cl treated sample of same portion of sliced beet- roots. Overall, the decline in betacyanin was lit- tle higher than betaxanthin in the outer tissues whereas opposite trend was found in the inner tissues. At the end of the storage, total beta- lain content was almost similar in SPC-TW and PSC-TW treated samples as well as in SPC-Cl and PSC-Cl treated samples of both tissues (Ta- ble 2). Samples of PCS treatments showed the lowest amount of total betalain contents at the end of storage in both tissues. It appears that although the declining rate of betalain was low- er in PCS method than other methods, the total amount of betalain content was comparative- ly lower than other methods at the end of stor- age probably due to the higher loss of betalain in this method on washing day. In this study, sanitization and washing favor larger pigment losses of beetroots slices owing to their expo- sure to water or sanitized solution and there- fore, we observed significant variation in beta- lain contents on washing day among the pro- cessing/sanitization methods. Similar to our results, the variations and decreases in beta- lains were also found in few studies (VITTI et al., 2005; KLUGE et al., 2006; VITTI et al., 2011). In contrast our pigment decline rate, LOPEZ OS- ORNIO and CHAVES (1998) found about 40-50% decreases in betalain amount in grated beet- root after 7 days of storage at 0°C, whereas at 4°C the decreases were greater. However, these authors did not measure the pigment contents at different tissues of beetroots, which we did in the present study. Betalains accumulate in cell vacuoles of the leaves, flowers and fruits of the plants that synthesize them, mainly in epi- dermal and/or subepidermal tissues (JACKMAN and SMITH, 1996). Moreover, it was reported that the total phenolic contents and the main betacyanin present in red beetroots distributed mostly towards the outer parts of the root and decreasing in the order peel, crown and flesh (KUJALA et al., 2000). This localized distribu- tion of betalains was also found in our study. However, due to large number of samples, we did not use the middle part of beetroots tissues and only the outer and inner tissues were used for biochemical measurement. Nevertheless, we assume that our fresh-cut beetroots slices were larger in size than that of grated beetroot which might prevented higher decline of pig- ments compared to that of LOPEZ OSORNIO and CHAVES (1998) findings. NILSON (1973) observed the contents of betacyanin and betaxanthin is cultivar dependent and the levels of these pig- ments are around 45 to 210 and 20 to 140 mg 100 g-1, respectively. Total phenolic contents Total phenolic contents of sliced beetroots (Fig. 2) followed similar distribution trend at different tissues that we found in betalain con- tents and is supported by the results of KUJALA et al. (2000). On the washing day, total phenolic contents also followed similar pattern as we ob- served for betalains contents among the treat- ments. The amount of total phenolic contents in the outer and inner tissues of beetroots samples ranged from 102.4 to 116.4 mg GAE 100 g-1 FW and 52.1 to 80.0 mg GAE 100 g-1 FW, respectively on washing day (Fig. 2). Higher amount of total phenolic content was observed in SPC method followed by PSC and PCS methods. However, we did not find any specific trend in total phenolic content of sliced beetroots during storage at low temperature. In the outer tissue, total phenol- ic contents declined on the third day of storage and then slightly increased the content or keep constant throughout the storage. At the end of the storage, total phenolic contents of this por- tion slightly increased (P>0.05) than the levels observed on washing day. PCS-TW treated sam- ple showed the lowest amount of total phenolic contents both on washing day and throughout the storage. At the end of storage, SPC-TW treat- ed sample showed the highest amount of total phenolic content (118.1 mg GAE 100 g-1 FW) fol- lowed by PSC-Cl treated sample (116.8 mg GAE 100 g-1 FW). Variations in total phenolic con- tents were also observed in the inner tissues on washing day and the contents either increased or decreased or maintained its level after fluctu- ating in between the storage. Although the high- est amount of total phenol (79.9 mg GAE 100 g-1 FW) was determined in SPC-TW treated inner tissues’ sample on washing day, PSC-Cl treated sample retained the highest amount (71.3 mg GAE 100 g-1 FW) among the treatments at the end of storage (Fig. 2). KUJALA et al. (2000) ob- served decreased amount of total phenolic con- tent until 63 days and the amount remained al- most unchanged until 196 days of storage of raw beetroots. The effects of different storage tem- peratures on the total phenolic content in plants have been studied and, like our study, both in- creases and decreases in phenolic contents have been reported. For example, LEWIS et al. (1999) observed an increase in total phenolic contents of colored potato tubers during storage at 4°C, whereas CORDENUNSI et al. (2005) found that total phenolic contents of strawberry remained 216 Ital. J. Food Sci., vol. 27 - 2015 Fig. 2 - Total phenol content of outer (above) and inner (below) tissues of fresh-cut red beetroots after processing with differ- ent sanitation methods and during storage at 5°C. Legends: same as shown in Fig. 1. constant or even slightly decreased during stor- age at different temperatures. Microbial quality Figure 3 shows the changes in total aerobic bacteria (TAB) count and yeast and mold (YM) count of fresh-cut red beetroots on washing day and during successive storage at low tempera- ture. Significant variation (P<0.05) was found in TAB among the washing treatments both on washing day and throughout the storage. The range of TAB was found 2.6 log CFU g-1 in PSC- Cl to 3.4 log CFU g-1 in SPC-TW treated samples on washing day. This result indicates that sani- tation of beetroots with 100 ppm chlorinated wa- ter ensured a significant reduction in TAB both in PSC and PCS methods. In order to reduce the microbial population, chlorine solution has been used as sanitizer in many fresh-cut vege- tables including beetroots (LOPEZ OSORNIO and CHAVES, 1997; VITTI et al., 2011) and sweet pota- toes (ERTURK and PICHA, 2006). However, stud- ies on microbial safety or monitoring of microbi- al population in fresh cut beetroots are still lim- ited as compared to other fresh produces. Since beetroots contain several bioactive compounds, its use as fresh-cut produce is promising where microbial safety should be addressed and en- sured. Beetroots washed with tap water either before or after peeling had nearly similar num- ber of TAB which indicates the necessity of san- itation of fresh-cut beetroots with appropriate sanitizer. Sanitation with chlorinated water be- Ital. J. Food Sci., vol. 27 - 2015 217 fore peeling showed almost no effect in reducing TAB as compared to after peeling or after slicing sanitation. ERTURK and PICHA (2006) reported that chlorination of sweet potatoes before slic- ing could not ensure acceptable microbiological quality of fresh-cut sweet potatoes. However, in our study we found significant reduction in TAB when beetroot samples were sanitized after peel- ing but before slicing though the numbers of TAB were significantly lower in after slicing sanitation throughout the storage (Fig. 3). LOPEZ OSORNIO and CHAVES (1997) found significant reduction in yeast and total aerobic count in grated beet- roots after washing treatment with chlorinated water. The number of TAB gradually increased until the end of storage with few fluctuations. Among the treatments, PCS-Cl treated sample showed the lowest numbers of TAB throughout the storage followed by PSC-Cl treated sample (Fig. 3). However, we found higher losses of beta- lain (Table 1), lower level of total phenol contents especially in inner tissues (Fig. 2) as well as low- er visual quality score (later discussion) in PCS- Cl treated samples which limited the potentiali- ty of this treatment. All tap water washed sam- ples regardless of processing methods showed similar pattern of changes in TAB. Sanitation of beetroot with 100 ppm chlorine water follow- ing SPC method exhibited similar number of TAB until 7 days of storage and the number in- creased thereafter to reach the similar number with PSC-TW treatment. Yeast and mold (YM) count, on the other hand, showed different patterns of changes that we ob- Fig. 3 - Effects of different sanitation methods on total aerobic bacteria (TAB) count and yeast and mold (YM) count of fresh- cut red beetroots during storage at 5°C. Legends: same as shown in Fig. 1. 218 Ital. J. Food Sci., vol. 27 - 2015 served in TAB (Fig. 3). Among the treatments, PSC-Cl treatment ensured the lowest number of YM count on the washing day (1.1 log CFU g-1) and throughout the storage. This result indicates that about 1.1 log CFU g-1 reduction in YM count was occurred in PSC-Cl treatment on the washing day as compared to PSC-TW treatment in which the highest YM count (2.2 log CFU g-1) was ob- served on that day. Unlike TAB count, there was no remarkable increase found in YM count when storage progressed. It appeared that survival and growth pattern of aerobic bacteria as well as yeast and mold are different in fresh cut beetroots. The decontamination effect of chlorinated water de- pends on dipping time, concentration of active chlorine, water temperature and the type of pro- duce used (ERTURK and PICHA, 2006; VITTI et al., 2011). In agreement with our results, ERTURK and PICHA (2006) found no significant reduction in yeast and mold count in fresh-cut sweet po- tato among control, chlorination of peeled whole roots and chlorination after slicing the roots re- gardless of water temperatures and concentration of chlorine, except for a significant reduction in chlorination after slicing at 20°C using 200 ppm chlorine solution. However, these authors found significant reduction in initial mesophilic popu- lation using both 100 and 200 ppm chlorine wa- ter washing of peeled whole sweet potato roots. LOPEZ OSORNIO and CHAVES (1997), on the oth- er hand, found significant decrease in the ini- tial yeast counts in grated beetroots after wash- ing in 252 ppm active chlorine solution at 8°C. These findings may suggest that higher concen- tration of active chlorine solution might be effec- tive in reducing yeast and mold count in root veg- etables. However, higher concentration of chlo- rine and longer dipping time caused a substan- tial decline in pigment contents in beetroots (VITTI et al., 2011). Our YM count result suggests that higher surface area exposed to washing treat- ment were more susceptible to YM contamina- tion and thereby unlikely TAB count, we found significantly (P<0.05) higher levels of YM count in PCS-Cl treatment (Fig. 3). Among the three sani- tation methods used in our study, peeling-sanita- tion-cutting (PSC) was the best method for reduc- ing YM count both on washing day and through- out the storage. Sensory quality Visual quality of sliced beetroot decreased gradually when storage time elapsed (Fig. 4). However, we did not notice off-odor in any sam- ple until the end of storage (data not shown). Although all samples retained marketable lim- it until 7 days of storage, samples processed only with SPC and PSC methods retained their marketable limit which was set at 6 in a 9-point scale until 10 days of storage. In a previous study, VITTI et al. (2005) also reported that min- imally processed beetroots could be stored un- til 10 days at low temperature and the produce quality drastically reduced corresponding with the increases in storage temperatures. Due to Fig. 4 - Visual quality scores of fresh-cut red beetroots during storage at 5°C after processing with different sanitation meth- ods. Legends: same as shown in Fig. 1. Ital. J. Food Sci., vol. 27 - 2015 219 the earlier development of whitish substances on the surface of beetroots slices, samples pro- cessed with PCS method received lower visual quality scores from 7 days of storage. Moreo- ver, we found higher losses of betalain and as a consequence higher values of whiteness in- dex in samples treated with this method (Ta- ble 2 and Fig. 1). However, the chlorine treat- ed sample of PCS methods showed the lowest number of TAB among the treatments (Fig. 3). This result indicates that maintenance of visual quality of fresh-cut beetroots may not depend on microbial load at least until certain limit. In general, consumer can only assess the sensory appearance. Hence, there is an increasing de- mand for the development of improved meth- ods that guarantee a high produce quality and safety until the end of shelf life, especially for fresh-cut produces. At the end of storage, sam- ples of all treatments exhibited the lower val- ues of marketable limit. Overall our results in- dicate that fresh-cut beetroots processed with SPC and PSC methods could be marketable un- til 10 days of storage at low temperature where- as samples processed with PCS could have only 7 days of marketable life. CONCLUSIONS Significantly lower values of chroma and higher values of whiteness index were found in samples processed with peeling – cutting –sani- tation (PCS) method, especially in PCS-Cl treat- ment. Although PCS-Cl treatment showed the lowest values of TAB throughout the storage, higher betalain decline along with lower level of total phenol and visual quality score limit- ed the storability of beetroots in this treatment only until 7 days. Beetroots processed with SPC method exhibited the highest amount of beta- lain and total phenol contents, but showed higher number of microbial population. Sam- ples of PSC method exhibited intermediate lev- els of betalain and PSC-Cl treatment ensured minimum number of microbial population. 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