Using Fluorescence Spectroscopy To Identify Milk from Grass-Fed

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Using fluorescence spectroscopy to identify milk from grass-fed dairy cows and to monitor its photodegradation Ujjal Bhattacharjee, Danielle Jarashow, Thomas A. Casey, Jacob W. Petrich, and Mark A. Rasmussen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05287 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Journal of Agricultural and Food Chemistry

1 2

Using fluorescence spectroscopy to identify milk from grass-fed

3

dairy cows and to monitor its photodegradation.

4 5 6 7

Ujjal Bhattacharjee,1,2 Danielle Jarashow,1 Thomas A. Casey,3

8

Jacob W. Petrich*1,2, and Mark A. Rasmussen*4

9 10 11

1

Department of Chemistry, Iowa State University, Ames, IA, USA.

12

2

U.S. Department of Energy Ames Laboratory, Ames, IA, USA.

13 14

3

Visiting scientist, Department of Chemistry, Iowa State University, Ames, IA, USA.

4

Leopold Center for Sustainable Agriculture, Iowa State University, Ames, IA, USA

15 16

* Corresponding authors.

17 18 19 20 21 22 23 24 1 ACS Paragon Plus Environment


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Abstract

27

Owing to its high omega-3 fatty acid content, milk from grass-fed dairy cows is becoming

28

increasingly more attractive to consumers. Consequently, it is important to identify the origins

29

of such products and to measure their content, at least relative to some standard. To date,

30

chromatography has been the most extensively used technique. Sample preparation and cost,

31

however, often reduce its wide-spread applicability.

32

fluorescence spectroscopy for such quantification by measuring the amount of chlorophyll

33

metabolites in the sample. Their content is significantly higher for milk from grass-fed cows as

34

compared to milk from grain/silage-fed cows. It is 0.11 ─ 0.13 µM in milk samples from grass-

35

fed cows; whereas, in milk from cows fed grain/silage rations, the concentration was 0.01─ 0.04

36

µM. In various organic-milk samples, the chlorophyll metabolite concentration was in the range

37

of 0.07─0.09 µM. In addition, we explored the mechanisms of photodegradation of milk.

38

Riboflavin and chlorophyll metabolites act as photosensitizers in milk for type-I and type-II

39

reactions, respectively. It was also observed that the presence of high levels of chlorophyll

40

metabolites can synergistically degrade riboflavin, contributing to the degradation of milk

41

quality.

Here, we report the effectiveness of

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Introduction

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Milk obtained from grass-fed cows is reported by some to be healthier than that from

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grain/silage-fed cows.1 In regions where fish is not a staple food, milk from grass-fed cows is an

47

alternative source for omega-3 fatty acids, which are beneficial for the prevention of cardio-

48

vascular diseases and hyperlipidemia.2,

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eicosapentaenoic acid and the ratio of eicosapentaenoic acid to arachidonic acid is significantly

50

higher in milk obtained from cows fed fresh green forage compared to that from cows fed

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harvested, preserved, and stored forage, such as corn silage.1 In spite of the reported benefits of

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milk from grass-fed cows, there has been very little research on developing analytical methods to

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confirm the authenticity of products labelled as milk from grass-fed cows. To date, the primary

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technique has been capillary gas-liquid chromatography, which quantifies fatty acid methyl

55

esters.1-6 In spite of the thoroughness of the process this is extremely inconvenient or impossible

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to implement for millions of pounds of milk produced every day.

3

The concentrations of linolenic acid and

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Fluorescence spectroscopy, however, is a powerful analytical tool; and we have

58

previously exploited it in a wide range of applications: detecting fecal and ingesta contamination

59

on beef carcasses7; detecting central nervous system tissue8; and monitoring the presence of

60

transmissible spongiform encephalopathies in sheep and cows.9,

10

We have also used

61

fluorescence

of

pheophorbide-a

62

pyropheophorbide-a (chlorophyll metabolites), which act as efflux pump inhibitors in bacteria.11

63

The work on both the detection of fecal and ingesta contamination and the study of the efflux

64

pump inhibitors specifically exploits the fluorescence of chlorophyll metabolites. In all such

65

established methods the principal motivation is developing a tool for fast and high-throughput

66

identification where exact chemical identity of the fluorophore or its absolute molar

spectroscopy

to

determine

the

concentrations


and


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concentration is less significant though in a relative scale it has been discussed. Recently,

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Andersen and Mortensen have proposed the use of fluorescence spectroscopy as a tool for

69

analyzing dairy products.12 Namely, chlorophyll or chlorophyll metabolites have been shown to

70

be present in variable amounts in almost all milk products, along with the predominant

71

fluorophore, riboflavin.

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Here we demonstrate that fluorescence spectroscopy can be used as a quantitative

73

measure of the relative content of chlorophyll metabolites in milk from grass-fed cows in a scale

74

established by fluorescence intensity calibration in methanol. Furthermore, we demonstrate that

75

the photodegradation of milk is dependent on the relative concentrations of riboflavin and

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chlorophyll metabolites.

77

Experimental methods

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Milk samples. Processed milk samples from several commercial brands were obtained

79

from local grocery stores. A minimum of 6 samples were obtained for each type of milk over a

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period of six months to take into account any variance in milk production. We have given these

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samples the generic labels A through E. All the experiments with milk were performed after

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homogenization. The samples from which cream was separated were not homogenized; and the

83

milk from these samples was not used for any experiments. Measurements were repeated at least

84

three times with fresh samples previously unexposed to light for each milk sample.

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characteristics of each sample are as follows: milk A (organic, whole, from grass-fed cows,

86

homogenized); milk B (conventional, from grain/silage-fed cows, whole, homogenized); milk D

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(whole, organic, homogenized, diet of cows unspecified). Fresh unprocessed milk samples were

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also obtained at the time of milking from cows at the Iowa State University (milk F) dairy and

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from an Iowa organic, pasture-based dairy farm (milk G). Separate cream samples for A, B, C


The

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(organic, from grass-fed cows), and E (organic, from grass-fed cows) were purchased from the

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same or different commercially available sources. The ISU (F) cows were fed a diet containing

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grain and ensiled forage, i.e., corn silage. The ISU cows did not have access to fresh pasture

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forage. The Iowa organic dairy farm (G) cows were grazed on pasture with about 85% of their

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diet coming from this fresh forage. The raw-milk spectra were obtained by homogenization with

95

a BeadBug homogenizer (Benchmark Scientific).

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Front-faced fluorescence spectroscopy.

Steady-state fluorescence spectra were

97

obtained with a SPEX Fluoromax-4 (ISA Jobin-Yvon/SPEX, Edison, NJ) with a 5-nm band-pass

98

for both excitation and emission and corrected for lamp spectral intensity and detector response.

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The samples (stored in the dark) were maintained in a front-faced orientation in a 1-mm cuvette.

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In order to excite chlorophyll and its metabolites preferentially over riboflavin, an excitation

101

wavelength of 420 nm was chosen, and a 420-nm interference filter was placed in the beam to

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ensure its spectral purity. Emission was collected at wavelengths greater than 450 nm using a

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cutoff filter. In order to reduce contributions from scattered light, crossed polarizers were placed

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before and after the sample.

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Calibration curve. In order to quantify the amount of chlorophyll metabolites in the

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milk samples, a calibration curve was constructed from pheophorbide-a in methanol at different

107

concentrations using the same instrument settings as were used for collecting the milk spectra.

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In addition, concentration range of pheophorbide-a was selected so that the corresponding

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fluorescence intensity fell in the same range as that observed for the milk samples. Recognizing

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the possibility that the fluorescence quantum yield of pheophorbide-a or chlorophyll metabolites

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may vary to a small extent from methanol to milk, we provide the concentration of chlorophyll

112

metabolites on a relative scale. Finally, to take into account any spectral shift between the milk



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samples and the pheophorbide-a calibration standard, we do not use peak intensities, but rather,

114

integrated areas of the spectra.

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Spectral processing. In order to quantify accurately the amount of chlorophyll

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metabolites in the milk spectrum, it is necessary to obtain the metabolite spectrum after

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subtracting the riboflavin spectrum in milk. The resulting difference spectra were described by a

118

linear combination of three spectra, which is a well-established method13, 14 for determining an

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individual spectrum from a mixture of fluorophores, where the fluorophores do not perturb each

120

other.15, 16 The difference fluorescence spectra can be written as: ‫ܫ‬௧௢௧ ሺߣሻ = ‫ܣ‬௣ ‫ܫ‬௣ ሺߣሻ + ‫ܣ‬௖ଶ ‫ܫ‬௖ଶ ሺߣሻ+‫ܣ‬௖ଷ ‫ܫ‬௖ଷ ሺߣሻ

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(1)

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Ip,, Ic2, and Ic3 are the spectra of pheophorbide-a and two other components in milk,

123

respectively (see SI equations 1, 2 and 3). The A’s are the weighted amplitudes of these spectra.

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Ip is well described by a sum of two lognormal lineshapes,17,

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pheophorbide-a fluorescence spectrum in methanol. Furthermore, λmax of Ip was found to be

126

same as that in methanol. Two other lognormal lineshapes are used to describe the other

127

components: Ic2 (λmax = 630 nm), attributed to protoporphyrin, which likely originates from

128

endogenous hemoglobin, myoglobin, and cytochrome C) 19, 20; and Ic3 (λmax = 588 nm), attributed

129

to endogenous flavin adenine dinucleotide (FAD)21, 22. In fitting the spectra, all the parameters

130

(see SI equations 1, 2 and 3) were kept constant except the weights of the individual profiles, Ap ,

131

Ac2, Ac3.

18

as is apparent from the

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Photodegradation studies. Photodegradation studies were performed with ambient,

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oxygenated samples and with degassed samples. Degassing was performed by means of freeze-

134

pump-thaw technique.23, 24 Six cycles of freeze-pump-thaw were used for all the samples. The

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milk was kept in a 1-cm cuvette exposed to a collimated beam of 470- or 650-nm light of known 6 ACS Paragon Plus Environment

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irradiance for a given time. Fluorescence spectra were taken with λex = 420 nm for different

137

exposure times.

138

Results and Discussion

139

Determination of chlorophyll and chlorophyll metabolite concentration. Andersen

140

and Mortensen have documented the fluorophores present in dairy products.12

141

appears to be predominant in the emission spectra with a maximum at 525 nm. Figure 1a

142

presents representative spectra of milk samples from commercial milk samples A, B and D,

143

normalized to the maximum of the riboflavin band. Figure 1b presents the difference spectra of

144

the milk and pure riboflavin. The spectra in Figure 1b shows presence of pheophorbide a11 (a

145

chlorophyll metabolite), providing a maximum at ~675 nm and a shoulder at ~720 nm. The data

146

indicate that the chlorophyll metabolite content is significantly higher in milk from grass-fed

147

cows (milk A) or organic milk (D) as compared to milk (B) from cows fed a conventional

148

grain/silage diet. From the prominence of the 675-nm peak in even the raw spectrum, one can

149

confidently assign the milk category. This assignment can be made on the basis of the ratio of

150

the peak intensity at 675 nm to that at 530 nm: 0.18-0.20, 0.09-0.12, and 0.14-0.18 for A, B and

151

D, respectively. A derivative spectrum also shows significant difference between milk from a

152

grass diet and a grain/silage.25

Riboflavin

153

To determine the concentration of chlorophyll metabolites, we first deconvoluted the

154

difference spectra into components as described above in order to obtain a spectrum from only

155

the chlorophyll metabolites.

156

components are explicitly shown in Figure S1. We constructed a calibration curve with

157

pheophorbide-a (Figure 2a and 2b), which yielded a slope of 1.61×107/µM. Integrated intensity

158

was used to eliminate the contribution from a small change in spectral lineshape, although the

The representation of the difference spectra in terms of the



159

peak maximum remains the same in methanol and in milk. The concentration of chlorophyll

160

metabolites (from samples in Figure 1) was found to be 0.11─0.13 µM in the milk sample from

161

grass-fed cows, whereas in milk from grain/silage-fed cows, the concentration was 0.01─0.04

162

µM. In organic-milk samples, the chlorophyll metabolite concentration was in the range of

163

0.07─0.09 µM. Thus chlorophyll concentration in milk from grass-fed cows is 4─10 times

164

higher than in milk from grain/silage-fed cows.

165

The ratio of the intensity of the maximum of the chlorophyll metabolite band to that of

166

the riboflavin band is much higher in cream than in milk (Figure 3 and 4). This is because the

167

chlorophyll metabolite partitions more favorably into a hydrophobic environment, which is the

168

result of the high lipid content of cream. Thus, we conclude it is very important to homogenize

169

the milk samples or standardize a milk processing procedure prior to fluorescence measurement.

170

The higher content of chlorophyll metabolites in cream from grass-fed cows (milk A, C and E)

171

than that from cows fed grain/silage (milk F) was also evident. In addition, we verified that

172

freezing the sample did not change the observed spectra, which is essential to determine if

173

samples are to be stored over long periods of time before analysis (see Figure S2).

174

Photodegradation studies. Photodegradation occurs by means of a photosensitizer, a

175

substance, which upon photoexcitation induces decomposition independent of oxygen

176

concentration (type I mechanism) or interacts with oxygen to generate the reactive species,

177

singlet oxygen (type II mechanism). Wold et al. demonstrated that photodegradation of milk can

178

be correlated with the intensity of the riboflavin band.19, 26-29 They cite six photosensitizers26:

179

riboflavin; and five molecules related to chlorophyll--protoporphyrin, hematoporphyrin,

180

chlorophyll-a, and two tetrapyrroles. This is consistent with our spectra presented in Figure 1.

181

Consequently, for our photodegradation studies, we investigated the potential of riboflavin and


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chlorophyll metabolites to degrade the sample. Samples were irradiated with ~50 mW/cm2 at

183

470 ± 5 nm, which excites the riboflavin predominantly, or at 650 ± 20 nm, which excites only

184


185

chlorophyll metabolites to sample degradation can be distinguished. If a fluorophore acts as a

186

photosensitizer, its emission intensity will decrease; and this decrease will be proportional to the

187

concentration of photosensitizers.30 Here we monitored the maxima of the fluorescence spectra

188

of riboflavin (λem ~ 530 nm) and of chlorophyll metabolites (λem ~ 675 nm). Experiments were

189

performed in the presence and the absence of oxygen, on milk samples from both conventional,

190

preserved forage-fed cows (milk B) and grass-fed cows (milk A), exciting either the riboflavin or

191


In this way, the relative contributions of riboflavin and the

192

When riboflavin is excited, the concentration of the chlorophyll metabolite signal

193

decreases when exposed to air (Figure 5). After degassing, there is no significant decrease in

194

chlorophyll concentration upon riboflavin excitation. This implicates a type-II photoreaction,

195

where the photosensitizer (riboflavin) produces reactive oxygen species. The data obtained does

196

not indicate a type-I reaction with a photobleaching rate independent of oxygen concentration.

197

However, the rate of bleaching (i.e., photodegradation) of riboflavin is similar in the presence

198

and absence of oxygen, suggesting a predominantly type-I photoreaction. Furthermore, the

199

photoproducts of riboflavin oxidation are visible as a broad band centered on ~500 nm. Similar

200

results were obtained for samples from conventional grain/corn silage-fed cows, milk B (Figure

201

S3).

202

Exposure to light at 650 nm, where only chlorophyll absorbs, bleaches chlorophyll

203

metabolites faster in the aerated than in the degassed sample, thus suggesting the type-II reaction,

204

as is shown in Figure 6a for milk A from grass-fed cows. Milk sample B from cows fed a



205

conserved forage diet yields a result similar to that of milk A (Figure S4). The peak intensity of

206

riboflavin in the milk A showed a faster decay in the degassed sample than in the aerated sample

207

(Figure 6b). On the other hand, for λex = 650 nm, the riboflavin intensity does not change in

208

milk B and hence has a lower amount of chlorophyll metabolites than milk A. Thus we suggest

209

that the larger quantity of chlorophyll metabolites enhances the degradation of riboflavin in milk

210

A.

211

photosensitized chlorophyll metabolites reacted with oxygen more efficiently. To understand the

212

mechanism of such synergistic degeneration of riboflavin and chlorophyll metabolites, further

213

studies are needed. The possible mechanisms can either be direct interaction such as electron

214

transfer with photoactivated chlorophyll metabolites

215

present in milk.

This is more apparent in the absence of oxygen, since in presence of oxygen the

31

or reaction via some other component

216

Lastly, in the Figures 5 and 6, the data points at the longest time periods are obtained

217

after the sample had been keep in the dark since the previous measurement. This is done in order

218

to obtain an estimate of the reversibility of the photodamage to the pigments. Both the

219

chlorophyll metabolites and riboflavin show a small amount of fluorescence recovery. This

220

suggests the possibility of a “dark state” in addition to the state responsible for permanent

221

bleaching. The degree of dark state varies with intensity and exposure time of the exciting light.

222

Such recovery has been observed in fluorescent proteins.32, 33

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Conclusions

224

We have demonstrated a potentially useful method, using fluorescence spectroscopy, to

225

determine the concentration of chlorophyll metabolites in a calibration scale in milk and thereby

226

establish a relationship between diet and the amount of chlorophyll metabolites present in the

227

milk. This method provides a simple and inexpensive means of identifying milk from grass-fed

228

cows. Furthermore, the method does not require complicated sample preparation. (Additional 10 ACS Paragon Plus Environment

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work may be necessary for obtaining absolute amounts of the chlorophyll metabolites and for

230

accounting for small variations in the measurements of the fluorescence quantum yields.) We

231

also discuss the need to keep the sample in complete darkness and refrigerated before testing

232

since exposure to light can alter the photoactive chlorophyll metabolite concentration, which is

233

represented by fluorescence peak intensity. Furthermore, we demonstrated the primary types of

234

photoreaction of the photosensitizers present: riboflavin and chlorophyll metabolites, which

235

were found to be type-I and type-II respectively. Additionally we observed that the presence of

236

chlorophyll metabolites can synergistically degrade riboflavin, which may necessitate more

237

stringent storage condition for grass-milk, although elucidating the exact mechanism of this

238

process requires further investigation.

239

Acknowledgments

240

We thank Dr. Francis Thicke, Radiance Dairy, Fairfield, Iowa, for graciously supplying samples

241

and Professor Leo Timms, Department of Animal Science, ISU, for assistance with collection of

242

samples at the ISU Dairy.

243

providing special-project seed funding for this work.

244

Supporting Information

245

Spectral decomposition of milk A and milk B (Figure S1); Effect of freezing on milk

246

fluorescence (Figure S2); Photodegradation kinetics of milk B (from cows on regular ration)

247

upon exciting riboflavin (Figure S3) and chlorophyll (Figure S4).

We thank the Leopold Center for Sustainable Agriculture for

248 249 250 251 252



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253 254 255 256 257

riboflavin milk A milk B milk D

1.0 0.8 0.6 0.4

Pheophorbide-a milk A milk B milk D

(b) 0.20

Intensity

Normalized Intensity

0.25 1.2 (a)

0.15 0.10 0.05

0.2

0.00

0.0 500

550

600

650

700

550

750

650

700

750

Wavelength (nm)

Wavelength (nm)

258

600

259

Figure 1.

260

a. Representative front-faced fluorescence spectra of milk A (black), milk B (green), milk D

261

(blue), and excess riboflavin in grain-fed milk where the milk was pre-treated with 650 nm

262

excitation for ~15 hours to bleach the chlorophyll completely or at least to the level which falls

263

below sensitivity of our instrument (magenta). The predominant band whose maximum is at 525

264

nm arises from riboflavin.

265

b.

266

fluorescence spectrum of pheophorbide-a in methanol (red).

Riboflavin-subtracted spectra of milk A (black), milk B (green), milk D (blue) and

267



6

20 4

Intensity (× 10 )

(a)

268

0.004 µM

16

0.013 µM 0.04 µM

12

0.122 µM

8

0.36 µM

4 0 600

Integrated Intensity (× 10 )

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650

700

750

Wavelength (nm)

6

(b)

5 4 3 2 1 0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

[ Pheophorbide-a ](µM)

269

Figure 2. (a) Fluorescence spectra of pheophorbide-a in methanol. (b) Calibration curve

270

obtained from the intensity of the emission maximum of pheophorbide-a vs. concentration.

271




1.4

272 273

cream A cream E cream C cream B

1.2 1.0 0.8 0.6 0.4 0.2

0.0 450 500 550 600 650 700 750 800

Wavelength (nm)

274

Figure 3. Representative front-faced fluorescence spectra of different cream samples, cream A

275

(black), cream B (red), cream C (blue) and cream E (green).

276 277


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milk G cream G milk F

1.0 0.8 0.6 0.4 0.2 0.0

500 550 600 650 700 750

Wavelength (nm) 278 279

Figure 4. Front-faced fluorescence spectra of raw milk G (black), raw milk F (green), and

280

cream separated from milk G (red).

281



400000

(a)

aerated

Intensity

300000 200000 100000 0

0 min 3 min 10 min 25 min 50 min 105 min 210 min 390 min 750 min recovery (450 min)

500 550 600 650 700 750 800

Wavelength (nm)

282

500000 (b)

Intensity

400000 300000 200000 100000 0

degassed

0 min 5 min 15 min 30 min 60 min 120 min 240 min 510 min 780 min recovery (420 min)

500 550 600 650 700 750 800

Wavelength (nm)

283 284

Figure 5.

285

Fluorescence spectra of milk A at different exposures to λex = 470 nm: a, aerated; b, degassed.

286

The intensity of the maxima of the riboflavin and chlorophyll bands at different exposure times:

287

aꞌ, aerated; bꞌ, degassed. Riboflavin maxima are denoted by the black dots (●); chlorophyll

288

metabolite maxima, by the red dots (●). In panels aꞌ and bꞌ, the data points at the longest times

289

are obtained after the sample had been keep in the dark since the previous measurement. This is

290

done in order to obtain an estimate of the reversibility of the photodamage to the pigments.

291


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1.2

292

1.0 0.8

chlorophyll metabolites aerated degassed

0.6 0.4 0.2 0.0

0

1.2 (b) riboflavin



(a)

200 400 600 800 1000 1200

Exposure Time (min)

1.0 0.8 0.6 0.4

aerated degassed

0.2 0.0

0

200 400 600 800 1000 1200

Exposure Time (min)

293

Figure 6. The intensity of the maxima of the emission bands of (a) chlorophyll metabolites and

294

of (b) riboflavin in milk A after different exposure times to 650 nm, aerated (●) and degassed

295

(●). In panel b, the data points at the longest times are obtained after the sample had been keep

296

in the dark since the previous measurement. This is done in order to obtain an estimate of the

297

reversibility of the photodamage to the pigments.

298 299 300



301 302

References

303 304

1.

Hebeisen, D. F.; Hoeflin, F.; Reusch, H. P.; Junker, E.; Lauterburg, B. H., Increased

305

concentrations of omega-3 fatty acids in milk and platelet rich plasma of grass-fed cows. Int J

306

Vitam Nutr Res. 1993, 63, 229-233.

307

2.

308

318, 549-557.

309

3.

310

between fish consumption and 20-year mortality from coronary heart disease. N. Engl. J. Med.

311

1985, 312, 1205-1208.

312

4.

313

3244-3257.

314

5.

315

Lipid Res. 1982, 21, 1-45.

316

6.

317

content of milk from cows fed different diets. J Dairy Sci. 1999, 82, 2146-2156.

318

7.

319

Fluorescence of dietary porphyrins as a basis for real-time detection of fecal contamination on

320

meat. J. Agric. Food Chem. 2003, 51, 3502-3507.

321

8.

322

Maistrovich, F. D.; Hamir, A. N.; Kehrli, M. J.; Richt, J.; Petrich, J. W., Fluorescence-based

323

method, exploiting lipofuscin, for real-time detection of central nervous system tissues on bovine

324

carcasses. J. Agric. Food Chem. 2008, 56, 6220–6226.

Leaf, A.; Weber, P. C., Cardiovascular effects of n-3 fatty acids. N. Engl. J. Med. 1988,

Kromhout, D.; Boschieter, E. B.; De Lezenne Coulander, C., The inverse relation

Grummer, R. R., Effect of feed on the composition of milk fat. J Dairy Sci. 1991, 74,

Tinoco, J., Dietary requirements and functions of alpha-linolenic acid in animals. Prog.

Dhiman, T. R.; Anand, G. R.; Satter, L. D.; Pariza, M. W., Conjugated linoleic acid

Ashby, K. D.; Wen, J.; Chowdhury, P.; Casey, T. A.; Rasmussen, M. A.; Petrich, J. W.,

Schönenbrücher, H.; Adhikary, R.; Mukherjee, P.; Casey, T. A.; Rasmussen, M. A.;


Page 18 of 22

Page 19 of 22


325

9.

Adhikary, R.; Mukherjee, P.; Krishnamoorthy, G.; Kunkle, R. A.; Casey, T. A.;

326

Rasmussen, M. A.; Petrich Jacob, W., Fluorescence spectroscopy of retina for diagnosis of

327

transmissible spongiform encephalopathies. Anal. Chem. 2010, 82, 4097-4101.

328

10.

329

Petrich, J. W., Fluorescence spectroscopy of the retina for the screening of bovine spongiform

330

encephalopathy. J. Agric. Food Chem. 2016, 64, 320–325.

331

11.

332

concentration of potential efflux pump inhibitors, pheophorbide a and pyropheophorbide a, in the

333

feces of animals by fluorescence spectroscopy. J. Agric. Food Chem. 2012, 60, 10456−10460.

334

12.

335

dairy products. J. Agric. Food Chem. 2008, 56, 720-729.

336

13.

337

spectrometry. Anal. Chem. 1980, 52, 351–352.

338

14.

Gans, P., Data fitting in the chemical sciences. Wiley: Leeds, UK, 1992.

339

15.

Satzger, H.; Spörlein, S.; Root, C.; Wachtveitl, J.; Zinth, W.; Gilch, P., Fluorescence

340

spectra of trans- and cis-azobenzene – emission from the franck–condon state. Chemical Physics

341

Letters 2003, 372, 216-223.

342

16.

343

Photoinduced trans-to-cis phase transition of polycrystalline azobenzene at low irradiance occurs

344

in the solid state. Chem. Phys. Chem. 2017, 18, 2526–2532.

345

17.

346

measurements of solvation dynamics. Chem. Phys. 1994, 183, 235–247.

Bhattacharjee, U.; Graham, C.; Czub, S.; Dudas, S.; Rasmussen, M. A.; Casey, T. A.;

Barnes, C. A.; Rasmussen, S. L.; Petrich, J. W.; Rasmussen, M. A., Determination of the

Andersen, C. M.; Mortensen, G., Fluorescence spectroscopy: A rapid tool for analyzing

Gans, P.; Gill, J. B., Comments on the critical evaluation of curve fitting in infrared

Bhattacharjee, U.; Freppon, D.; Men, L.; Vela, J.; Smith, E. A.; Petrich, J. W.,

Fee, R. S.; Maroncelli, M., Estimating the time-zero spectrum in time-resolved emmsion



347

18.

Subhash, N.; Mohanan, C. N., Curve-fit analysis of chlorophyll fluorescence spectra:

348

Application to nutrient stress detection in sunflower. Remote Sens. Environ. 1997, 60, 347-356.

349

19.

350

occurring chlorophyll and porphyrins in light-induced oxidation of dairy products. A study based

351

on fluorescence spectroscopy and sensory analysis. Int. Dairy J. 2005, 15, 343–353.

352

20.

353

of normal mouse skin exposed to 5-aminolaevulinic acid and red light. J. Photochem. Photobiol.

354

B 2001, 61, 78–86.

355

21.

356

allows the characterization of mild heat treatments applied to milk. Relations with the

357

denaturation of milk proteins. J. Agric. Food Chem. 2005, 53, 502-507.

358

22.

359

cells using redox ratio and double excitation wavelengths autofluorescence. Biomed. Opt.

360

Express 2015, 6, 977-986.

361

23.

362

ion (nbt2+) in degassed aqueous solution. Photochem. Photobiol. 1983, 38, 137-140.

363

24.

364

12, 382-384.

365

25.

366

1982, 19, 22-46.

367

26.

368

photosensitizers in butter detected by fluorescence spectroscopy and multivariate curve

369

resolution. J. Agric. Food Chem. 2006, 54, 10197–10204.

Wold, J. P.; Veberg, A.; Nilsen, A.; Iani, V.; Juzenas, P.; Moan, J., The role of naturally

Juzenas, P.; Iani, V.; Bagdonas, S.; Rotomskis, R.; Moan, J., Fluorescence spectroscopy

Kulmyrzaev, A. A.; Levieux, D.; Dufour, E. R., Front-face fluorescence spectroscopy

Palmer, S.; Litvinova, K.; Rafailov, E. U.; Nabi, G., Detection of urinary bladder cancer

Takakubo, M.; Faure, J., Riboflavin sensitized photoreduction of nitro blue tetrazolium

Gilroy, D.; Mayne, J. E. O., The de-aeration of aqueous solutions. J. Appl.Chem. 1962,

O'Haver, T. C., Derivative spectroscopy and its applications in analysis. Anal. Proc.

Wold, J. P.; Bro, R.; Veberg, A.; Lundby, F.; Nielsen, A. N.; Moan, J., Active


Page 20 of 22

Page 21 of 22


370

27.

Wold, J. P.; Mielnik, M., Nondestructive assessment of lipid oxidation in minced poultry

371

meat by autofluorescence spectroscopy. J. Food Sci. 2000, 65, 87-95.

372

28.

373

rancidity in complex meat products by front face fluorescence spectroscopy. J. Food Sci. 2002,

374

67, 2397-2404.

375

29.

376

and color of light on photooxidation in cheese: A study based on sensory analysis and

377

fluorescence spectroscopy. Int. Dairy J. 2006, 16, 1218-1226.

378

30.

379

Enviorn. Pathol. Toxicol. Oncol. 2006, 25, 7-28.

380

31.

381

chlorophyll-a and quinones in solution ii. Some effects of non-quinoid donors and acceptors:

382

Riboflavins, thioctic acid and nadh. Biochim. Biophis. Acta 1962, 66, 308-318.

383

32.

384

F., Real-time light-driven dynamics of the fluorescence emission in single green fluorescent

385

protein molecules. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7237–7242.

386

33.

387

switching behaviour of single molecules of green fluorescent protein. Nature 1997, 388, 355-

388

358.

Wold, J. P.; Mielnik, M.; Pettersen, M. K.; Aaby, K.; Baardseth, P., Rapid assessment of

Wold, J. P.; Veberg, A.; Lundby, F.; Nilsen, A. N.; Moan, J., Influence of storage time

Juzeniene, A.; Nielsen, K. P.; Moan, J., Biophysical aspects of photodynamic therapy. J.

Tollin, G.; Green, G., Light-induced single electron-transfer reactions between

Garcia-Parajo, M. F.; Segers-Nolten, G. M. J.; Veerman, J.-A.; Greve, J.; van Hulst, N.

Dickson, R. M.; Cubitt, A. B.; Tsien, R. Y.; Moerner, W. E., On/off blinking and

389

390

391



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Using Fluorescence Spectroscopy To Identify Milk from Grass-Fed

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