Citrus Unshiu Marc.) During the Maturation Period

horticulturae

Article Monitoring of Fluorescence Characteristics of Satsuma Mandarin (Citrus unshiu Marc.) during the Maturation Period

Muharﬁza 1,2,*, Firmanda Al-Riza Dimas 1,3, Yoshito Saito 1, Kenta Itakura 1, Yasushi Kohno 4, Tetsuhito Suzuki 1, Makoto Kuramoto 5 and Naoshi Kondo 1

1 Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan; dimasﬁ[email protected] (F.A.-R.D.); [email protected] (Y.S.); [email protected] (K.I.); [email protected] (T.S.); [email protected] (N.K.) 2 Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture, Jakarta 12540, Indonesia 3 Department of Agricultural Engineering, Faculty of Agricultural Technology, University of Brawijaya, Jl. Veteran, Malang 65145, Indonesia 4 Department of Planning and Environment, Ehime Research Institute of Agriculture, Forestry and Fisheries, Matsuyama, Ehime 700-2405, Japan; [email protected] 5 Advanced Research Support Center, Ehime University, 2-5 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan; [email protected] * Correspondence: muharﬁ[email protected]; Tel.: +81-80-4973-1979

Academic Editor: Douglas D. Archbold Received: 20 July 2017; Accepted: 17 October 2017; Published: 25 October 2017

Abstract: Monitoring the maturation process of Satsuma mandarin (Citrus unshiu Marc.) by determining the soluble solids (SS) and acid content non-destructively is needed. Fluorescence components potentially offer such means of accessing fruit maturity characteristics in the orchard. The aim of this study was to determine the potential of fluorescence spectroscopy for monitoring the stage of citrus maturity. Four major fluorescent components in peel and/or flesh were found including chlorophyll-a (excitation (Ex) 410 nm, emission (Em) 675 nm) and chlorophyll-b (Ex 460 nm, Em 650 nm),polymethoxyflavones (PMFs) (Ex 260 nm and 370 nm, Em 540 nm), coumarin (Ex 330 nm, Em 400 nm), and a tryptophan-like compound (Ex 260 nm, Em 330 nm). Our results indicated a significant (R2 = 0.9554) logarithmic ratio between tryptophan-like compoundsExEm and chlorophyll-aExEm with the SS:acid ratio. Also, the log of the ratio of PMFs from the peel (ExExEm was significantly correlated with the SS:acid ratio (R2 = 0.8207). While the latter correlation was not as strong as the former, it does demonstrate the opportunity to develop a non-destructive field measurement of fluorescent peel compounds as an indirect index of fruit maturity.

Keywords: ﬂuorescence; maturity index; soluble solid and acid ratio; chlorophyll-a; chlorophyll-b; polymethoxyﬂavones; tryptophan-like

1. Introduction After bananas and apples, citrus has been the third most popular fruit consumed in Japan since 1975. Among the various citrus cultivars grown in Japan, Satsuma mandarin (Citrus unshiu Marc.) is one of the most popular. Around 90 percent of Satsuma mandarin produced in Japan is consumed fresh, 7 percent is juice, and 3 percent is canned [1]. To provide high-quality citrus, an accurate estimation of optimum maturity for harvest is required. If over-mature citrus are harvested, they are more susceptible to mechanical damage during handling, which can lead to abundant rotten fruit. On the other hand, harvesting immature fruit will result in unmarketable products, with unpleasant

Horticulturae 2017, 3, 51; doi:10.3390/horticulturae3040051 www.mdpi.com/journal/horticulturae Horticulturae 2017, 3, 51 2 of 11

flavor, since the fruit is not yet physiologically mature. Thus, maturity index is an important quality parameter for determining the time of harvest. As noted above, the most important factor determining fruit quality is maturity [2], defined as the phase of development leading to the attainment of physiological or horticultural maturity. In other words, maturity is the final stage of natural growth and development [3]. In this respect, a maturity index can quantify whether a certain product is mature, providing the fruit grading industry with a trade rule or trading strategy, as well as efficient use of resources [3]. There are several parameters commonly used to determine maturity, such as color change [4]; soluble solids (SS)-to-acid ratio [5]; and firmness, size, and shape [3,6]. Among these parameters, the SS:acid ratio is the most important since it determines the flavor of the products. Conventionally, farmers determine citrus maturity by physical inspection, observing color and SS content measurements, or chronologically (calculation of growth period since post-anthesis period). However, observations of color, or through calendar calculations, are subjective and inaccurate methods of determining maturity. Furthermore, SS and acid content measurements are destructive, can only be applied to a few samples, and are labor-intensive. Considerable efforts have gone into developing alternative maturity detection methods, such as the electronic nose, machine vision systems, and visible-near infrared (VIS-NIR). One such successful method for monitoring mandarin ripeness and quality attributes (firmness, SS, and acidity) is use of an electronic nose technique [7]. However, this technique requires strict environmental controls due to sensitivity to humidity, and is expensive to implement. Furthermore, a machine vision system for maturity determination based on color information and the ratio of total SS to titratable acidity (TA) has been developed [8], but requires complicated artificial lighting in a closely controlled dark room. Portable VIS-NIR spectroscopy has also been successfully developed to estimate mandarin maturity status based on total SS and (TA) [9]. Like the other methods however, this method is relatively expensive. In contrast, fluorescence spectroscopy, a sensitive, cheap, fast, and easily applied technique [10], has the potential to detect information from rich fluorophore dynamics in order to monitor changes in plant tissue compounds. Fruit maturity in Satsuma mandarin may be related to the medium fluorescence emissions [11], which are relatively easy to detect. Recently, fluorescence spectroscopy has been used to indicate the maturity of fruits such as apples and grapes by using a multiplex sensor [12,13]. To the best our knowledge, however, there is no research investigating the potential of fluorescence techniques for maturity determination in citrus. Thus, the aim of this research was to investigate the potential of fluorescence spectroscopy for estimating the stage of maturity in Satsuma mandarin. The fluorescence characteristics were monitored during the growth and maturation stages (stage II and stage III) and compared with a standard maturity index (SS/acid ratio).

2. Materials and Methods

2.1. Materials At each monthly sampling time, 22 Satsuma mandarins (cv. Miyagawa Wase) located at the top of the canopy (exposed to sunlight) were harvested from June 2016 (30 days after anthesis) to December 2016 (210 days after anthesis) from 35-year-old trees grown in the Ehime Research Plant orchard, Ehime Prefecture, Japan.

2.2. Methods

2.2.1. Sample Preparation The harvested fruit were sorted manually based on peel appearance, wherein undamaged fruit were chosen. A color image of these fruit was captured using a color camera equipped with a polarized ﬁlter and illuminated by four halogen lamps. From these 22 samples, based on color value (red/green) Horticulturae 2017, 3, 51 3 of 11

Horticulturae 2017, 3, 51 3 of 11 ratio (Python 2.7.12 with OpenCV library), 10 fruit were selected to minimize variability (those fruit replication. All fruit chosen for subsequent measurement were cleaned using an ultrasonic cleaner with the closest R/G ratios). The 10 selected fruit were divided into ﬁve groups for data replication. (As-One US-2A, As-One, Osaka, Japan) for 5–10 min to remove dust and dirt, then wiped dry and All fruit chosen for subsequent measurement were cleaned using an ultrasonic cleaner (As-One US-2A, kept at room temperature (25 °C) overnight in order to reach equilibrium temperature before As-One, Osaka, Japan) for 5–10 min to remove dust and dirt, then wiped dry and kept at room extraction. temperature (25 ◦C) overnight in order to reach equilibrium temperature before extraction.

2.2.2. Peel and Flesh Extraction Sampled fruit fruit were were divided divided into into two two main main parts: parts: peel peel (including (including albedo albedo and flavedo) and flavedo) and flesh and (includingflesh (including juice sacs juice and sacs segments), and segments), as shown as shownin Figu inre Figure1. Citrus1. Citruspeel contains peel contains a number a number of volatile of volatileoils [14]. oils These [14]. volatile These volatile oils can oils be caneasily be easilydissolv dissolveded in organic in organic solvents solvents [11]. [In11 this]. In experiment, this experiment, the fluorescentthe fluorescent substances substances were were extracted extracted using using chloroform chloroform as as a solvent a solvent (Wako (Wako Pure Pure Chemical Chemical Industries, Industries, Osaka, Japan).

Figure 1. Diagrammatic equatorial cross-section of citrus fruit [[15].15].

For June and July samples, since the peel and fleshflesh could not be separated, the whole fruit was extracted. The crushed citrus was weighed (Shima (Shimadzudzu AUW-220D, Shimadzu Co., Kyoto, Japan) and mixed withwith chloroformchloroform atat a a ratio ratio of of 1:3 1:3 (1 (1 g g by by 3 mL3 mL chloroform), chloroform), then then homogenized homogenized and and macerated macerated for for60 min 60 min using using a mechanical a mechanical shaker shaker (Wakenyaku (Wakenyaku 2240, Wakenbtech 2240, Wakenbtech Co., Ltd., Co., Kyoto, Ltd., Japan). Kyoto, This Japan). extracted This extractedfluorescence fluorescence compounds. compounds. Afterward, Afterward, liquid extracts liquid were extracts filtered were (Whatman filtered No. (Whatman 41) and placed No. 41) directly and placedinto a vial. directly June into and a July vial. data June for and the July peel data and fo fleshr the were peel referred and flesh to were as the referred peel). to as the peel). For the August to December samples, the peel wa wass separated from the flesh. flesh. For For peel peel extraction, extraction, four pieces of peel with an areaarea ofof 100100 squaresquare mmmm (10(10 mmmm ×× 1010 mm) mm) from from different locations around the bottom part of the the fruit fruit were were sampled, sampled, weighed, weighed, and and their their thickness thickness was was measured measured using using a a Vernier Vernier caliper (Mitutoyo PCX-15, Mitutoyo Co., Co., Kanagawa, Kanagawa, Ja Japan).pan). Peel Peel and and flesh flesh were were extracted extracted separately. separately. Peel samples were cut into small segments using scissorsscissors and mixed with chloroform at a ratio of 1:5 (1 unit areaarea byby 55 mLmL chloroform).chloroform). FleshwasFleshwas also also cut cut into into small small segments segments and and mixed mixed at at a a ratio ratio of of 1:3 1:3 (1 (1 g gby by 3 3 mL mL chloroform). chloroform). Then, Then, both both were were separately separately maceratedmacerated andand homogenizedhomogenized for 60 min, filtered, filtered, and extracts placed directly into a vial.

2.2.3. Soluble Solid Content and Acidity Measurement Brix, which is sugar or SS content, and acids (acidity) are the most important quality parameters used to represent the sweetnesssweetness andand acidityacidity ofof freshfresh citruscitrus fruit.fruit. TheThe ratioratio ofof SSSS toto acidityacidity (SS/acid)(SS/acid) is widely used as a maturitymaturity criterion for non-climacteric fruit. SS and acidity were measured using a hand-held digital refractometer (Atago PAL-BX PAL-BX|ACID│ACID F5, Atago Co., Ltd., Tokyo, Japan). From the fleshflesh of each sample, juice was squeezed by hand and filteredfiltered (Whatman No. 41 filter filter paper). Then, a 0.2-mL clear liquid aliquot of the filtered filtered juice wa wass directly pipetted onto the sensor and SS content (% Brix) measured. Once the Brix value was recorded, the sample was diluted by adding distilled water—up to 10 mL—on the sensor, and the acidity was measured.

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Horticulturae 2017, 3, 51 4 of 11 (% Brix) measured. Once the Brix value was recorded, the sample was diluted by adding distilled water—up to 10 mL—on the sensor, and the acidity was measured. 2.2.4. Fluorescence Spectrum Measurement 2.2.4.Fluorescence Fluorescence measurements Spectrum Measurement were conducted sequentially the following day. Liquid samples wereFluorescence placed into four measurements clear fluorescence were conducted quartz glass sequentially cuvettes the with following a 10-mm day. path Liquid length, samples and weretheir placedspectra intowere four measured. clear fluorescence For this spectral quartz glassmeasurem cuvettesent, witha spectrofluorophotometer a 10-mm path length, and (Jasco their FP-8300, spectra wereJasco measured.Co., Tokyo, For Japan) this spectralwas used measurement, to scan the exci a spectrofluorophotometertation (Ex) and emission (Jasco (Em) FP-8300,wavelengths. Jasco The Co., TM Tokyo,instrument Japan) was was connected used to scan to thea computer, excitation wh (Ex)ich and had emission special (Em) software wavelengths. (SpectraManager The instrument Tokyo, was Japan)connected for spectra to a computer, capture whichand analysis. had special The excitation software (SpectraManager(Ex) wavelength TMwasTokyo, scanned Japan) from for 200 spectra to 550 nmcapture at 10-nm and increments analysis. The and excitation the emission (Ex) (Em) wavelength wavelength was was scanned scanned from from 200 210 to to 550 750 nm nm at at 10-nm 1-nm increments.increments andThe theexcitation emission and (Em) emission wavelength slits were was scannedmaintained from at 210 5 nm, to 750 with nm a atscan 1-nm speed increments. of 5000 Thenm/min excitation and response and emission time of slits 50 ms were for maintainedall measurements. at 5 nm, with a scan speed of 5000 nm/min and response time of 50 ms for all measurements. 2.2.5. Data Processing 2.2.5.All Data raw Processing spectrum results were captured and corrected by Rhodamine B and a halogen light sourceAll using raw spectrumSpectraManager results wereTM application captured software, and corrected after by which Rhodamine the intensity B and was a halogen normalized light source using theusing Raman SpectraManager scatter peakTM ofapplication water [16]. software,Finally, the after peak which finder the in intensity the SpectraManager was normalizedTM application using the softwareRaman scatter was peakused of to water find [ 16desirable]. Finally, fluorescence the peak finder intensity in the SpectraManager peaks. The statisticalTM application analyses software were wasconducted used to on find averaged desirable raw fluorescence data using intensity Microsoft peaks. Excel The 2013. statistical A t test analyses used, and were the conducted significance on thresholdaveraged rawwas dataset at using 0.001. Microsoft Excel 2013. A t test used, and the significance threshold was set at 0.001. 3. Results and Discussion 3. Results and Discussion 3.1. Fruit Growth and Development 3.1. Fruit Growth and Development Citrus fruit growth and development follows a typical sigmoid growth curve, divided into three stagesCitrus [17], as fruit shown growth in Figure and development2. The first stage follows involves a typical rapid cell sigmoid division growth and then curve, a period divided of slow into growth.three stages The [growth17], as shownis controlled in Figure by 2indole. The firstacetic stage acid involves (IAA) and rapid indoleacetamide cell division and(IAM) then [18]. a periodPhysiological of slow fruit growth. abscission The growth also occurs is controlled through by indolethis period, acetic acidwhich (IAA) is caused and indoleacetamide by hormonal (IAM)imbalance [18]. [19]. Physiological During this fruit stage, abscission peel tissue also occursundergoes through intensive this period, changes which with is production caused by hormonalof a large numberimbalance of [cells,19]. During causing this the stage, peel volume peel tissue to be undergoes much greater intensive than changes that of the with pulp. production Moreover, of a citrus large juicenumber in this ofcells, period causing is exiguous the peel and volume the activities to bemuch of the greaterenzymic than systems that ofand the chlorophyll pulp. Moreover, content citrus also abundantjuice in this [20]. period After is exiguousreaching anda peak, the activitiesthe peel ofthic thekness enzymic diminishes systems in and proportion chlorophyll to contentthe internal also tissue.abundant [20]. After reaching a peak, the peel thickness diminishes in proportion to the internal tissue.

Figure 2. Growth and development stages of Satsuma mandarin in Japan.

In stage II, rapid cell enlargement takes place as the fruit enlarges physically and liquid begins In stage II, rapid cell enlargement takes place as the fruit enlarges physically and liquid begins to accumulate. This cell enlargement is commonly promoted by auxins [5], controlling fruit size. to accumulate. This cell enlargement is commonly promoted by auxins [5], controlling fruit size. During this stage, the volume of juice sacs rises, developing their distinctive liquids. This liquid During this stage, the volume of juice sacs rises, developing their distinctive liquids. This liquid component is high in organic acids and low in SS; the main organic acid in citrus juice is citric acid. component is high in organic acids and low in SS; the main organic acid in citrus juice is citric acid. Furthermore, citric acid is generated in the mitochondria of juice cells by the Krebs cycle and stored in the vacuoles [15]. At the beginning of liquid accumulation, the high citric acid concentration is one of the reasons for the resulting low SS/acid ratio. During fruit growth th e ratioincreases due to catabolism of citric acid, while SS content increases due to the high amount of soluble carbohydrates. In this stage, the

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Furthermore,Horticulturae citric acid 2017, 3 is, x FOR generated PEER REVIEW in the mitochondria of juice cells by the Krebs cycle5 of 11 and stored in the vacuoles [15]. At the beginningIn stage ofII, rapid liquid cell accumulation, enlargement takes the place high as the citric fruit acidenlarges concentration physically and isliquid one begins of the reasons for to accumulate. This cell enlargement is commonly promoted by auxins [5], controlling fruit size. the resultingDuring low SS/acidthis stage, ratio. the volume During of juice fruit sacs growth rises, dev theloping e ratioincreases their distinctive due liquids. to catabolism This liquid of citric acid, while SS contentcomponent increases is high in due organic to the acids high and low amount in SS; the of main soluble organic carbohydrates. acid in citrus juice In is citric this acid stage,. the citrus fruit peel undergoesFurthermore, a citric color acid change is generated from in greenthe mitochondria to yellow of juice or orange—a cells by the Krebs process cycle and that stored is characterized in the vacuoles [15]. by the progressiveAt the loss beginning of chlorophyll, of liquid accumulation, which is the accompanied high citric acid byconcentration formation is one of otherof the reasons pigments such as anthocyaninsfor orthe carotenoids.resulting low SS/acid The ratio. color During conversion fruit growth results th e ratio fromincreases the differentiationdue to catabolism of of citric chloroplasts to chromoplasts,acid, and while is SS influenced content increases by many due to factors, the high includingamount of soluble hormones, carbohydrates. environmental In this stage, conditions,the and citrus fruit peel undergoes a color change from green to yellow or orange—a process that is nutrient availability.characterized Moreover, by the progressive exogenous loss of ethylene chlorophyll, accelerates which is accompanied chlorophyll by formation degradation of other and increases chlorophyllasepigment activitys such as [21 anthocyanins]. This natural or carotenoids. color change The color is conversion associated results with from the the accumulationdifferentiation of sugars and the degradationof chloroplasts of acids to chromoplasts, [22]. and is influenced by many factors, including hormones, environmental conditions, and nutrient availability. Moreover, exogenous ethylene accelerates Finally,chlorophyll stage III degradation or the maturing and increases period chlorophyllase starts around activity 5 [21] months. This natural after thecolor startchange of is fruit growth. In this period,associated growth with is the mostly accumulation complete of sugars and and fruit the ripeningdegradation is of a acids non-climacteric [22]. process. This process is economicallyFinally, important stage III since or the thematuring color period of citrus starts around fruit is 5 months a crucial after quality the start of parameter fruit growth. for In consumers. this period, growth is mostly complete and fruit ripening is a non-climacteric process. This process is During thiseconomically process, Satsumaimportant since color the becomescolor of citrus orange, fruit is agrowth crucial quality is minimized, parameter for andconsumers. metabolic shifts occur to integrateDuring this biochemical process, Satsuma and color physiological becomes orange, changesgrowth is minimized that eventually, and metabolic render shifts occur the flesh edible. Farmers harvestto integrate the fruit biochemical a onth and to physiological 6 weeks later, changes in earlythat eventually to mid-November, render the flesh atedible the. Farmers optimum maturity harvest the fruit a onth to 6 weeks later, in early to mid-November, at the optimum maturity stage stage based on SS content determined by random sampling. After harvest, the maturing process will based on SS content determined by random sampling. After harvest, the maturing process will cease, cease, accompaniedaccompanied by by respiration, respiration, major major changes changes in flavor in, and flavor, senescence and senescence.. Our resultsOur demonstrated results demonstrate thatd in that November, in November the, the SS:acidSS:acid ratio ratio reached reached a maximum a maximum value (Figure value (Figure3). Furthermore,3). postponingFurthermore, harvestpostponing until harvest December until December did not did change not change the ratio.the ratio However,. However, a physiologicala physiological disorder such as puffing between peel and flesh may form and could result in disorder suchmechanical as puffing damage between while handling peel, andwhich flesh is caused may by formthe collision and between could resultfruit [23] in. mechanical damage while handling, which is caused by the collision between fruit [23].

Figure 3. RatioFigure of 3. Ratio soluble of soluble solid solid (SS)/acid (SS)/acid content content in juice in during juice the during growth the and growthmaturing stage ands.maturing Each stages. Each data pointdata point is the is the mean mean± ± S.D. of five ﬁve replications. replications.

3.2. Suspected Compound in Satsuma Mandarin (unshiu) Excitation and Emission Matrices (EEM) during 3.2. SuspectedMaturing Compound Period in Satsuma Mandarin (unshiu) Excitation and Emission Matrices (EEM) during Maturing PeriodStage II and III are critical periods in citrus quality determination, since citrus liquid accumulates in these stages. Therefore, this paper focuses on these two stages. The EEM data, shown in Figure 4, Stage IIillustrates and III the are six critical main peaks periods observed in citrusat the beginning quality of determination, water accumulation. since The citrusfirst and liquid second accumulates in these stages.peaks Therefore, are a1 (Ex 460 this nm and paper Em 650 focuses nm) and on a2 these (Ex 410 two nm and stages. Em 675 The nm) EEM for both data, the peel shown and in Figure4, illustrates theflesh, six which main is suspected peaks to observed be chlorophyll at (a1 the is a beginningchlorophyll-b and of watera2 is a chlorophyll accumulation.-a) [24]. The first and second peaks are a1 (Ex 460 nm and Em 650 nm) and a2 (Ex 410 nm and Em 675 nm) for both the peel and flesh, which is suspected to be chlorophyll (a1 is a chlorophyll-b and a2 is a chlorophyll-a) [24]. The third and fourth peaks are b1 (Ex 370 nm, Em 540 nm) and b2 (Ex 260 nm, Em 540 nm), which correspond to polymethoxyflavones (PMFs) [25]. PMFs are a subclass of flavonoids, which are richer in the peel than in the flesh. The fifth peak is c (Ex 330 nm, Em 400 nm); this peak corresponds to coumarin [26]. Coumarin is a secondary plant metabolite that is a native inhibitor, also known to be a plant growth regulator. Finally, the sixth peak is d (Ex 260 nm, Em 330 nm), identified by Sun et al. (2010) as being related to a tryptophan-like compound from Citrus aurantium L. [27]. HorticulturaeHorticulturae 20172017,, 33,, 5151 6 of 11

Figure 4.4. Fluorescence excitation and emission matric matriceses (EEM) of Satsuma mandarin during the maturing period from August to December: (A) peel in August; ( C) in December; ( B) flesh ﬂesh in August; ((DD)) inin December.December. TheThe colorcolor barsbars showshow thethe ﬂuorescentfluorescent intensityintensity inin RamanRaman units.units.

3.3. Time Series FluorescenceFluorescence duringduring thethe GrowthGrowth andand MaturingMaturing PeriodsPeriods Chlorophyll presentpresent mainly mainly in in the the peel peel can can be be visually visually identified identified (Figure (Figure2). In 2). contrast, In contrast, there there was nowas significant no significant level level of chlorophyll of chlorophyll detected detected in the in flesh. the flesh. During During maturation, maturation, chlorophyll chlorophyll degrades degrades with thewith alteration the alteration of chloroplasts of chloroplasts to chromoplasts, to chromoplasts, which is controlledwhich is controlled by internal andby internal external and factors, external such asfactors, plant such hormones as plant [28 hormones], SS, and temperature[28], SS, and [temperature29]. [29]. Chlorophyll in the peel significantlysignificantly decreased fromfrom June to September (Figure5 5)) andand slowlyslowly disappeared in October. TheThe degradationdegradation associated with color changechange revealedrevealed previouslypreviously maskedmasked pigments.pigments. This may be caused by the activity of chlorophyllase and chemical reactions [15]. [15]. Chlorophyll inin citrus consistsconsists ofof chlorophyll-a andand chlorophyll-b.chlorophyll-b. Chlorophyll-a is the most common typetype ofof chlorophyll,chlorophyll, predominantpredominant inin allall oxygen-evolvingoxygen-evolving photosyntheticphotosynthetic organisms.organisms. Furthermore, chlorophyll-a isis usedused inin foodfood processingprocessing as as an an appearance appearance control control agent agent for for colors. colors. Chlorophyll-b Chlorophyll-b is is a yellow-greena yellow-green chlorophyll, chlorophyll, and and its its function function is tois to absorb absorb blue blue light light energy. energy. PMFs are majormajor fluorescencefluorescence compoundscompounds in citrus;citrus; they are well-knownwell-known asas plantplant defensivedefensive components against pathogens. Moreover, Moreover, PMFs PMFs are are a a constituent constituent of of bitter bitter flavor flavor of citrus fruit, although they do not contribute to the taste of citruscitrus juice at their naturalnatural concentration range [30]. [30]. During the maturation period, PMFs in the peel sign significantlyificantly decreased from June to September, then theirtheir concentrationconcentration declineddeclined slightlyslightly untiluntil December. On the other hand, PMFs in the fleshflesh fluctuatedfluctuated unsteadily, decliningdeclining inin August,August, stabilizingstabilizing in September–October, andand suddenlysuddenly increasingincreasing againagain in November, whichwhich mightmight bebe causedcaused byby changeschanges inin acidacid content.content. In addition, the solubility of PMFs in thethe waterwater phasephase ofof biologicalbiological systemssystems increasesincreases withwith acidityacidity [[31].31]. The tryptophan-like compound compound appears appears in in both both tissues, tissues, with with both both showing showing similar similar patterns. patterns. The Thelevels levels of this of thiscompound compound slowly slowly increased increased from from June June toto reach reach a apeak peak at at harvest harvest (November), and decreased inin DecemberDecember in in the the over-mature over-mature stage. stage. In In plant plant tissue, tissue, tryptophan tryptophan is a is precursor a precursor to IAA to IAA and playsand plays a crucial a crucial role inrole plant in plant growth, growth, [32,33 [32,33].].

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Figure 5. Fluorescence compound dynamics by time on citruscitrus peel and flesh.flesh. Each data point is the mean ±± S.D.S.D. ofof five five replications. replications. No No coumarin coumarin was was detected detected in the in peel the and peel no and chlorophyll no chlorophyll was detected was indetected the flesh, in the so neitherflesh, so are neither shown. are shown.

Coumarins have signiﬁcantsignificant biological functions, such as chemoprevention against pathogens in plant tissue, regulation of abiotic stress, oxidativ oxidativee stress, and probably hormonal hormonal regulations regulations [34,35].[34,35]. As shownshown in in Figure Figure5, coumarin5, coumarin was onlywas observedonly observed in the ﬂesh.in the The flesh. lowest The intensity lowest wasintensity in September, was in whichSeptember, might which be caused might be by caused the hormonal by the hormonal regulation regulation of coumarin of coumarin itself, as itself, it could as it inhibitcould inhibit plant growthplant growth [36]. [36]. Therefore, Therefore, in September, in September, coumarin coumar wasin was present present with with a a weak weak intensity intensity asas thethe fruit was enteringentering thethe maturing maturing period, period, and and its its levels levels remained remained relatively relatively stable stable until until December, December, and while and. citruswhile diametercitrus diameter was relatively was relatively stable fromstable September from September until December. until December. The decrease in size observed from November to December in some data might be caused by postponing the harvest, which make makess the citrus more mature with a subsequent decline in quality; another reason could be the season turning to winterwinter and the resulting sudden low temperatures in the tree environment.

3.4. Soluble Solid and Acid MetabolismMetabolism during the Maturing Period Citrus juice contains SS measured as (% Brix) and acidity measured as a % citric acid. These are the main internal quality parameters that have commonlycommonly been used in many fields.fields. Furthermore, the ratio ratio of of Brix Brix to to acidity acidity in in the the fruit fruit juice juice is th ise themost most common common method method to estimate to estimate the maturity the maturity level levelof citrus of citrus fruit fruitand andis reflected is reflected in the in theflavor flavor [37] [37. More]. More than than 75% 75% of of the the total total SS SS is is sucrose, sucrose, which increases during fruit maturation on the tree (Figure 66)) andand isis furtherfurther utilizedutilized forfor thethe synthesissynthesis ofof various polysaccharides, including pectin.pectin. The decrease in acidity (mostly organic acid) is considered to be due to aa dilutiondilution effecteffect asas thethe fruitfruit increasesincreases inin sizesize andand accumulatesaccumulates water. Organic acids are respiratory substrates in the fruit; a high respiratory quotient indicates the utilization of acid through the tricarboxylic acidacid (TCA)(TCA) cycle, cycle, which which is oxidizedis oxidized and and produces produces ATPs ATPs (adenosine (adenosine triphosphates) triphosphates) for thefor synthesisthe synthesis of new of new compounds compounds [35]. The[35]. change The change of concentration of concentration of SS in of Satsuma SS in Satsuma mandarin mandarin was low fromwas low August from to August September, to September, but rapidly but increased rapidly inincreased October in and October reached and the reached maximum the inmaximum November. in November.

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Figure 6. Soluble solids (SS) and acidity (as % citric acid) during the growth and maturing stages. Figure 6. Soluble solids (SS) and acidity (as % citric acid) during the growth and maturing stages. 3.5. Potential Maturity Indices in Satsuma Mandarin 3.5. Potential Maturity Indices in Satsuma Mandarin Chlorophyll content is commonly used as a quality indicator in many fruits and vegetables. For example,Chlorophyll many content researchers is commonly have suggested used as a using quality measurement indicator in of many chlorophyll fruits and content vegetables. to evaluate For example,the quality many of grapes researchers and apples. have suggested They used using a ratio measurement of chlorophyll of chlorophyll a and b and content correlated to evaluate it with thephysical quality or chemicalof grapes properties. and apples The. They advantage used aof ratio this of method chlorophyll is the lower a and dependence b and correlated on orientation it with physicaland distance, or chemical and it hasproperties. a potential The to advantage be used as of a this future method measurement. is the lower Furthermore, dependence chlorophyll on orientation has andalso distance, been proposed and it as has an a internal potential quality to be parameter, used as aby future calculating measurement. the correlation Furthermore, between chlorophyll chlorophyll haswith also SS:acid been ratio proposed and fruit as firmnessan internal to evaluate quality theparameter, ripening by stage calculating in apples. the Those correlation had a quite between good chlorophyllcorrelation ofwith 0.699 SS:acid and 0.674,ratio and respectively fruit firmness [12]. to Moreover, evaluate the previous ripening researchers stage in apples. also succeeded Those had in apromoting quite good the correlation chlorophyll of fluorescence0.699 and 0.674, technique respectively as a non-destructive [12]. Moreover, evaluation previous forresearchers flavonols also and succeededanthocyanins in promoting in grapes [ 13the]. chlorophyll fluorescence technique as a non-destructive evaluation for flavonolsDuring and the anthocyanins maturing period, in grapes chlorophyll [13]. in the citrus peel gradually decreased. In the present work,During chlorophyll the maturing fluorescence period, intensity chlorophyll was compared in the citrus with peel the SS:acidgradually ratio. decreased. Our data In demonstrated the present work,that chlorophyll-a chlorophyll andfluorescence -b were weaklyintensity correlated was compared with the with SS:acid the ratio, SS:acidR2 =ratio. 0.441 Our and 0.474,data demonstratedrespectively. Furthermore, that chlorophyll-a the chlorophyll-a and -b were andweakly -b ratio correlated also showed with the a very SS:acid low ratio, correlation R2 = 0.441 with and the 0.474,SS:acid respectively. ratio (R2 = 0.0593). Furthermore, However, the citrus chlorophyll-a have slightly and different-b ratio also fluorescent showed compounds a very low compared correlation to withthose the in applesSS:acid andratio grapes, (R2 = 0.0593). creating However, an opportunity citrus tohave utilize slightly fluorescent different compounds fluorescent as compounds a maturity comparedindex A modofied to those in index apples from and the grapes, peel iscreating presented an opportunity because the to fluorescent utilize fluorescent peel compounds compounds could as aprovide maturity a convenient index A waymodofied to determine index thefrom quality the peel of citrus is presented quickly. because the fluorescent peel compoundsBesides could chlorophyll, provide the a convenient log of the ratio way of to PMFs determine was considerd the quality because of citrus they quickly. were more abundant in theBesides citrus peelchlorophyll, compared the to thelog flesh. of the In theratio present of PMFs results was (Figure considerd4), they because appeared they at two were different more abundantexcitation in wavelengths the citrus peel (260 compar nm anded 370to the nm) flesh. and In one the emission present results wavelength (Figure (540 4), nm).they appeared The ratio at of twothese different two wavelengths excitation potentiallywavelengths could (260 be nm used and as 370 an index;nm) and it isone dimensionless emission wavelength and an independent (540 nm). Themeasurement. ratio of these The two log wavelengths of the ratio between potentially two could PMFs be (Ex used 370 as nm, an Em index; 540 it nm is divideddimensionless by Ex 260and nm, an independentEm 540 nm) measurement. were derived, The and log a significant of the ratio linear between correlation two PMFs (P (Ex< 0.001) 370 nm, with Em SS 540 and nm acid divided ratio by(R 2Ex= 0.8207)260 nm, was Em found,540 nm) as were shown derived, in Figure and7 .a Insignificant August andlinear September correlation (the (P final < 0.001) growth with period), SS and acidthe citrusratio ( colorR2 = 0.8207) was mostly was greenfound, and as shown the citrus in Figure juice was 6. In acidic. August Then, and the September PMFs versus (the SS/acidfinal growth ratio period),gradually the increased citrus color during was the mostly maturing green period, and the stabilizing citrus juice at the was end acidic. of October. Then, the PMFs versus SS/acid ratio gradually increased during the maturing period, stabilizing at the end of October.

Horticulturae 2017, 3, 51 9 of 11 Horticulturae 2017, 3, 51 9 of 11 Horticulturae 2017, 3, 51 9 of 11

Figure 6. 7. CorrelationCorrelation between log of the ratio of polymethoxyflavones polymethoxyﬂavones (PMFs) (Ex 370 nm/Ex 260 260 nm) nm) Figure 6. Correlation between log of the ratio of polymethoxyflavones (PMFs) (Ex 370 nm/Ex 260 nm) and SS/acid ratio. ratio. and SS/acid ratio. Another possible index is the logarithmic ratio between tryptophan-like compound and AnotherAnother possible possible indexindex isis thethe logarithmiclogarithmic ratio between between tryptophan-like tryptophan-like compound compound and and chlorophyll-a, as shown in Figure 7. The logarithmic ratio exhibited a skewness towards high values, chlorophyll-a,chlorophyll-a, as as shown shown in in Figure Figure8 .7. The The logarithmiclogarithmic ratio exhibited a a skewness skewness towards towards high high values, values, which demonstrated multiplicative factors. There was a significant correlation (P < 0.001) with SS and whichwhich demonstrated demonstrated multiplicative multiplicative factors. factors. ThereThere was a significant signiﬁcant correlation correlation (P (P < <0.001) 0.001) with with SS SSand and acid content (R2 = 0.9554), where both the SS: acid ratio as well as the tryptophan-like compound to acidacid content content (R (2R2= = 0.9554), 0.9554), wherewhere both the SS: SS:acid acid ratio as as well well as as the the tryptophan-like tryptophan-like compound compound to to chlorophyll-a ratio increased during the maturing period. chlorophyll-chlorophyll-a ratioa ratio increased increased during during thethe maturingmaturing period.

FigureFigure 8. 7.Logarithmic Logarithmic correlation correlationbetween between thethe ratioratio of tryptophan-like/chlorophyll-a (Ex (Ex 260 260 nm nm and and Figure 7. Logarithmic correlation between the ratio of tryptophan-like/chlorophyll-a (Ex 260 nm and EmEm 330 330 nm nm divided divided Ex Ex 410 410 nm nm and and EmEm 675675 nm)nm) and SS/acid ratio. ratio. Em 330 nm divided Ex 410 nm and Em 675 nm) and SS/acid ratio.

4.4. Conclusions Conclusions 4. Conclusions FluorescenceFluorescence excitation-emission excitation-emission matrices matrices (EEM)(EEM) were used used in in this this study study to toidentify identify fluorescence fluorescence peaksFluorescence at Ex 410 nm excitation-emission and Em 675 nm suspected matrices (EEM)to be associated were used with in this chlorophyll-a, study to identify and fluorescence fluorescence peaks at Ex 410 nm and Em 675 nm suspected to be associated with chlorophyll-a, and fluorescence peakspeaks at atEx Ex 410 460 nm nm and and Em Em 675 650 nm nm suspected suspected toto bebe associated withwith chlorophyll-b. chlorophyll-a, The and compounds fluorescence peaks at Ex 460 nm and Em 650 nm suspected to be associated with chlorophyll-b. The compounds peakswhich at hadEx 460 peaks nm andat Ex Em 260 650 nm nmand suspected 370 nm and to Em be 540associated nm (in thewith peel) chlorophyll-b. and Ex 260 nm The and compounds Em 530 which had peaks at Ex 260 nm and 370 nm and Em 540 nm (in the peel) and Ex 260 nm and whichnm (inhad the peaksflesh) atcorresponded Ex 260 nm and to PMFs. 370 nm The and fluore Emscence 540 nm peak (in theat Ex peel) 330 and nm Exand 260 Em nm 380 and nm Em was 530 Em 530 nm (in the flesh) corresponded to PMFs. The fluorescence peak at Ex 330 nm and Em 380 nm nmsuspected (in the flesh) to be corresponded coumarin and, to PMFs.finally, Thethe fluorefluorescencescence peakpeak atat ExEx 330260 nm nm and and Em Em 380 330 nm nm was was suspected to be coumarin and, finally, the fluorescence peak at Ex 260 nm and Em 330 nm suspectedcorresponded to be tocoumarin tryptophan-like and, finally, compound. the fluo Serescenceveral index peak candidates at Ex 260 showednm and potential Em 330 as nm correspondedmaturity indices. toto tryptophan-like tryptophan-like These include compound. thecompound. logarithmic Several Seratioveral index between index candidates tryptophan-likecandidates showed showed potential compound potential as maturity and as maturityindices.chlorophyll-a, These indices. include which These were the inclu logarithmic significantlyde the logarithmic ratio correlated between ( Rratio2 tryptophan-like= 0.9554), between and tryptophan-likethe compoundlog of the ratio and compound between chlorophyll-a, two and 2 which were significantly correlated (R = 0.9554),2 and the log of the ratio between two PMFs, which chlorophyll-a,PMFs, which which was also were significantly significantly correlated correlated (R = ( R0.8207).2 = 0.9554), and the log of the ratio between two was also significantly correlated (R2 = 0.8207). PMFs, which was also significantly correlated (R2 = 0.8207). Acknowledgment: We would like to thank the Ministry of Agriculture of the Republic of Indonesia for granting Acknowledgments:the scholarship duringWe wouldthis study, like th toanks thank to theSupporting Ministry Program of Agriculture for InteRaction-Based of the Republic Initiative of Indonesia Team for Studies granting Acknowledgment: We would like to thank the Ministry of Agriculture of the Republic of Indonesia for granting the(SPIRITS) scholarship for funding during thisthis study,research, thanks and thanks to Supporting to Garry ProgramPiller for proof-reading for InteRaction-Based this manuscript. Initiative Team Studies (SPIRITS)the scholarship for funding during this this research, study, th andanks thanks to Supporting to Garry Program Piller for for proof-reading InteRaction-Based this manuscript. Initiative Team Studies (SPIRITS) for funding this research, and thanks to Garry Piller for proof-reading this manuscript. Author Contributions: N.K. was the project supervisor. M. designed the experiments presented in this paper, and performed the experiments and analysis with the assistant of F.A.-R.D., Y.S. and K.I., M. wrote and edited the manuscript with the assistance of F.A.-R.D., T.S., M.K., Y.S., and K.I.

Horticulturae 2017, 3, 51 10 of 11

Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Kurai, T. 2016 Japan Citrus Annual; USDA Foreign Agricultural Service: Tokyo, Japan, 2016. 2. Kader, A.A. Fruit maturity, ripening, and quality relationships. Acta Hortic. 1999, 57, 203–208. [CrossRef] 3. Reid, M.S. Definition of Maturity. Postharvest Technol. Hortic. Crops 2002, 3311, 55. 4. Cary, P.R. Citrus Fruit Maturity Vol-26; MPKV: Maharastra, India, 1974. 5. Iglesias, D.J.; Cercós, M.; Colmenero-Flores, J.M.; Naranjo, M.A.; Ríos, G.; Carrera, E.; Ruiz-Rivero, O.; Lliso, I.; Morillon, R.; Tadeo, F.R.; et al. Physiology of citrus fruiting. Braz. J. Plant Physiol. 2007, 19, 333–362. [CrossRef] 6. Crisosto, C.H. Stone fruit maturity indices: A descriptive. Postharvest News Inf. 1994, 5, 65N–68N. 7.G ómez, A.H.; Wang, J.; Pereira, A.G. Mandarin ripeness monitoring and quality attribute evaluation using an electronic nose technique. Trans. ASABE 2007, 50, 2137–2142. [CrossRef] 8. Yibin, Y.; Rao, X.; Ma, J. Methodology for Nondestructive Inspection of Citrus Maturity with Machine Vision–Transactions of The Chinese Society of Agricultural Engineering. Available online: http://en.cnki. com.cn/Article_en/CJFDTOTAL-NYGU200402034.htm (accessed on 6 April 2017). 9. Antonucci, F.; Pallottino, F.; Paglia, G.; Palma, A.; D’Aquino, S.; Menesatti, P. Non-destructive estimation of mandarin maturity Status through portable VIS-NIR spectrophotometer. Food Bioprocess Technol. 2011, 4, 809–813. [CrossRef] 10. Christensen, J.; Povlsen, V.T.; Sørensen, J. Application of Fluorescence Spectroscopy and Chemometrics in the Evaluation of Processed Cheese During Storage. J. Dairy Sci. 2003, 86, 1101–1107. [CrossRef] 11. Momin, M.A.; Kondo, N.; Kuramoto, M.; Ogawa, Y.; Yamamoto, K.; Shiigi, T. Investigation of excitation wavelength for fluorescence emission of citrus peels based on UV-VIS spectra. Eng. Agric. Environ. Food 2012, 5, 126–132. [CrossRef] 12. Betemps, D.L.; Fachinello, J.C.; Galarça, S.P.; Portela, N.M.; Remorini, D.; Massai, R.; Agati, G. Non-destructive evaluation of ripening and quality traits in apples using a multiparametric fluorescence sensor. J. Sci. Food Agric. 2012, 92, 1855–1864. [CrossRef][PubMed] 13. Ghozlen, N.B.; Cerovic, Z.G.; Germain, C.; Toutain, S.; Latouche, G. Non-destructive optical monitoring of grape maturation by proximal sensing. Sensors 2010, 10, 10040–10068. [CrossRef][PubMed] 14. Song, H.S.; Lan Phi, N.T.; Park, Y.-H.; Sawamura, M. Volatile profiles in cold-pressed peel oil from korean and japanese shiranui (Citrus unshiu Marcov. × C. sinensis Osbeck × C. reticulata Blanco). Biosci. Biotechnol. Biochem 2006, 70, 737–739. [CrossRef] [PubMed] 15. Seymour, G.B.; Taylor, J.E.; Tucker, G.A. Biochemistry of Fruit Ripening; Springer: Dordrecht, The Netherlands, 1993; ISBN 978-94-010-4689-3. 16. Lawaetz, A.J.; Stedmon, C.A. Fluorescence intensity calibration using the raman scatter peak of water. Appl. Spectrosc. 2009, 63, 936–940. [CrossRef][PubMed] 17. Bain, J.M. Morphological, anatomical, and physiological changes in the developing fruit of the Valencia orange, Citrus sinensis (L.) Osbeck. Aust. J. Bot. 1958, 6, 1–23. [CrossRef] 18. Igoshi, M.; Yamaguchi, I.; Takahashi, N.; Hirose, K. Plant growth substances in the young fruit of Citrus unshiu. Agric. Biol. Chem. 1971, 35, 629–631. [CrossRef] 19. Takahashi, N.; Yamaguchi, I.; Kono, T.; Igoshi, M.; Hirose, K.; Suzuki, K. Characterization of plant growth substances in Citrus unshiu and their change in fruit development. Plant Cell Physiol. 1975, 16, 1101–1111. [CrossRef] 20. Goren, R.; Monselise, S.P. Inter-relations of hesperidin, some other natural components and certain enzyme systems in developing shamouti orange fruits. J. Hortic. Sci. 1965, 40, 83–99. [CrossRef] 21. Fujii, H.; Shimada, T.; Sugiyama, A.; Nishikawa, F.; Endo, T.; Nakano, M.; Ikoma, Y.; Shimizu, T.; Omura, M. Profiling ethylene-responsive genes in mature mandarin fruit using a citrus 22K oligoarray. Plant Sci. 2007, 173, 340–348. [CrossRef] 22. Fidelibus, M.W.; Koch, K.E.; Davies, F.S. Gibberellic acid alters sucrose, hexoses, and their gradients in peel tissues during color break delay in “Hamlin” orange. J. Am. Soc. Hortic. Sci. 2008, 133, 760–767. 23. Agustí, M.; Martinez-Fuentes, A.; Mesejo, C. Citrus fruit quality. Physiological basis and techniques of improvement. Agrociencia 2002, 6, 1–16. Horticulturae 2017, 3, 51 11 of 11

24. Ustin, S.L.; Gitelson, A.A.; Jacquemoud, S.; Schaepman, M.; Asner, G.P.; Gamon, J.A.; Zarco-Tejada, P. Retrieval of foliar information about plant pigment systems from high resolution spectroscopy. Remote Sens. Environ. 2009, 113, S67–S77. [CrossRef] 25. Li, S.; Lo, C.-Y.; Ho, C.-T. Hydroxylated polymethoxyflavones and methylated flavonoids in sweet orange (Citrus sinensis) peel. J. Agric. Food Chem. 2006, 54, 4176–4185. [CrossRef][PubMed] 26. Fujii, K.; Iyi, N.; Hashizume, H.; Shimomura, S.; Ando, T. Preparation of integrated coumarin/cyanine systems within an interlayer of phyllosilicate and fluorescence resonance energy transfer. Chem. Mater. 2009, 21, 1179–1181. [CrossRef] 27. Sun, Y.; Zhang, H.; Sun, Y.; Zhang, Y.; Liu, H.; Cheng, J.; Bi, S.; Zhang, H. Study of interaction between protein and main active components in Citrus aurantium L. by optical spectroscopy. J. Lumin. 2010, 130, 270–279. [CrossRef] 28. El-Otmani, D.M.; Lovatt, C.J.; Jr, C.W.C.; Agustí, M. Plant growth regulators in citriculture: Factors regulating endogenous levels in Citrus tissues. Crit. Rev. Plant Sci. 1995, 14, 367–412. [CrossRef] 29. Takagi, T.; Mukai, H.; Ichikawa, T.; Suzuki, T. Effects of temperature and sugar accumulation in fruits on color development of satsuma mandarin. J. Jpn. Soc. Hortic. Sci. 1994, 62, 725–731. [CrossRef] 30. Veldhuis, M.K.; Swift, L.J.; Scott, W.C. Fully-methoxylated flavones in Florida orange juices. J. Agric. Food Chem. 1970, 18, 590–592. [CrossRef] 31. Musialik, M.; Kuzmicz, R.; Pawłowski, T.S.; Litwinienko, G. Acidity of hydroxyl groups: An overlooked Influence on antiradical properties of flavonoids. J. Org. Chem. 2009, 74, 2699–2709. [CrossRef][PubMed] 32. Murch, S.J.; KrishnaRaj, S.; Saxena, P.K. Tryptophan is a precursor for melatonin and serotonin biosynthesis in in vitro regenerated St. John’s wort (Hypericum perforatum L. cv. Anthos) plants. Plant Cell Rep. 2000, 19, 698–704. [CrossRef] 33. Radwanski, E.R.; Last, R.L. Tryptophan biosynthesis and metabolism: Biochemical and molecular genetics. Plant Cell 1995, 7, 921–934. [CrossRef][PubMed] 34. Gutierrez, M.-C.; Parry, A.; Tena, M.; Jorrin, J.; Edwards, R. Abiotic elicitation of coumarin phytoalexins in sunflower. Phytochemistry 1995, 38, 1185–1191. [CrossRef] 35. Ladaniya, M. Citrus Fruit: Biology, Technology and Evaluation; Academic Press: Croydon, UK, 2010; ISBN 978-0-08-055623-9. 36. Thimann, K.V.; Bonner, W.D. Inhibition of plant growth by protoanemonin and coumarin, and its prevention by BAL. Proc. Natl. Acad. Sci. USA 1949, 35, 272–276. [CrossRef][PubMed] 37. Olmo, M.; Nadas, A.; García, J.M. Nondestructive methods to evaluate maturity level of oranges. J. Food Sci. 2000, 65, 365–369. [CrossRef]

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