Indian black rice: A brewing raw material with novel functionality

Publisher Rights Statement: This is the peer reviewed version of the following article: Moirangthem, K., Jenkins, D., Ramakrishna, P., Rajkumari, R. and Cook, D. (2019). Indian black rice: A brewing raw material with novel functionality. Journal of the Institute of Brewing, which has been published in final form at https://doi.org/10.1002/jib.584. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of SelfArchived Versions.

ABSTRACT (250 words current 295) 1 Indian black rice (Chakhao Poireiton) is a pigmented variety, rich in anthocyanins and other 2 phytonutrients. With increasing interest in the use of local raw materials in brewing, it was of 3 interest to develop protocols for malting and brewing with Chakhao Poireiton to see in 4 particular whether the antioxidant capacity of anthocyanins could be delivered into finished 5 beer. Protocols for brewing with 100% malted rice were developed and the performance of 6 Indian black rice compared with that of an Italian white rice cultivar suited to brewing. The 7 apparent fermentabilities of rice worts were 69.5% (black) and 67.3% (white), yielding beers 8 of 3.28 and 3.19 % ABV respectively. Black rice worts were FAN deficient (83.5 mg/L relative 9 to 137 mg/L for white rice) and would need nitrogen supplementation to avoid issues with 10 fermentation, e.g. elevated diacetyl. Black rice beer had an orange-red hue as a result of 11 extraction of anthocyanin pigments (2.84 mg/L). The oxidative stability of 100% rice beers 12 was measured using Electron Spin Resonance (ESR) spectroscopy and both samples were 13 found to be unusually stable. Interestingly, when rice beers were blended with a control 14 barley malt derived lager in varying proportions (10, 25, 50%), the oxidative stability was 15 improved, relative to the control lager, particularly so in the case of black rice beer, which 16 contained an antioxidant capacity over and above that of the white rice beer. Future studies 17 are required to determine whether the noted oxidative stability of 100% rice malt beers 18 results in a more flavour stable beer. 19 20 KEYWORDS: Indian black rice, 100% rice beer, ESR, beer oxidative stability, beer 21 polyphenols. 22

INTRODUCTION 23
India is the largest rice producer in the world. With around 80% of this production utilized for 24 domestic consumption, it is also the largest consumer of rice 1 . The North -Eastern states of 25 India, such as Manipur, are home to a diverse range of traditional aromatic rice landraces 2 . 26 Once such variety, very popular in Manipur is the black rice -Chakhao Poireiton, belonging to 27 the species Oryza sativa L. indica. 28 29 Black rice appears black due to the presence anthocyanins, dark purple pigments, which are 30 present in its bran layer. Anthocyanins are antioxidants and the levels accumulated in black 31 rice bran are considered to be one of the highest levels found in foods 3 . Anthocyanins are 32 known for their ability to protect cells from damage due to biotic and abiotic stresses and 33 have also been considered as potential cancer chemo-preventative agents 4 . As a dietary 34 antioxidant, they can also help combat reactive oxygen species, free radicals and help 35 decrease the risk of chronic diseases such as coronary heart disease 5 . Additionally, they are 36 approved for use as a food additive or colouring agent in the EU, Australia and New Zealand 37 with the E number E163 (INS number 163) 6 . 38 39 In addition to antioxidants, black rice contains high amounts of flavonoid phytonutrients, 40 gamma oryzanol, polyphenols, Vitamin E, dietary fibre and minerals such as iron and copper. 41 It is a better source of plant based protein than normal white rice 7 . It is thus considered to be 42 a premium rice product from a nutritional perspective. There have been very limited studies 43 on black rice Chakhao Poireiton. It is a waxy rice and has been reported to be composed of 44 approximately 7% protein, 4% fat, 76% carbohydrate and 2% amylose with a gelatinisation 45 temperature between 75 and 92°C 8 . Figure 1 shows the paddy and different fractions of black 46 rice as it goes through an industrial milling process. However, black rice is usually not polished 47 in order to maintain its bran and the anthocyanin, giving the rice a chewy texture when 48 cooked.

50
In the brewing industry rice has been used as an adjunct mainly due to its neutral flavour. 51 However, brewing a 100% rice beer is somewhat more challenging. Its starch fails to undergo 52 complete saccharification during mashing. This has been reported to be attributed to the high 53 gelatinization temperature of its starch, insufficient starch-degrading enzymes in malted rice 54 and insufficient degradation of the structural protein of the endosperm cell wall needed prior 55 to or simultaneously with starch modification 9 . There have been only limited studies on the 56 production of rice beer. Usansa et al. reported that enzyme production during malting of rice 57 was dependent on the rice variety and did not correlate with amylose content 10 . There have 58 been reports of successful brewing with 100% rice malt, made possible by optimising the 59 mashing conditions 9 , and of the experimental development of speciality rice malts, roasted 60 to enhance flavour and the colour 11 . Amylase activity, needed for starch degradation, 61 depends greatly on the different incubation conditions. For rice, the minimum temperature 62 is 10-12°C, the optimal temperature is 30-37°C and the maximum temperature is 40-42°C 63 inside 12 . Additionally, optimum temperature conditions for malting black rice were reported 64 to be 30°C 13,14 , which is close to room temperature in Asian countries. Ceppi  with a Purospher STAR rp-18 end-capped column (250 x 4.6 mm, 3 µm particle size; Merck 128 Millipore, UK) coupled with a C18 guard cartridge from Phenomenex (UK). Peak areas 129 were extracted at 280 nm and total run time was 65 min. Samples were analysed in triplicate 130 and phenolic acid concentrations were determined from calibration curves generated from 131 external standards run at concentrations of 1, 10, 20, 40 mg/L.

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Beer analysis: The following parameters were measured in duplicate as for wort above: pH,

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Resonance (ESR) spectroscopy. 153 The oxidative stability of beers were assayed using a forcing test at 60°C during which the 154 time-course of free radical formation was measured using Electron Spin Resonance 155 spectroscopy (Bruker E-scan; Bruker Corporation, MA, USA) with N-tert-Butyl-α-156 phenylnitrone (PBN) as spin trap 18 . PBN (678 mg) was dissolved first in 500 µL of ethanol 157 (Fisher Scientific) and 500 µL water added. 280uL of PBN solution was added to each beer 158 sample (7 mL). Samples were placed in a 60°C heating block at 60 second intervals. ESR 159 spectra were recorded with a centre field of 3478 G and sweep width of 17 G. The microwave 160 bridge had a power of 2.31 mW and frequency of 9.77. Receiver gain was 1261, modulation 161 frequency 86 kHz, modulation amplitude 1.1 G, modulation phase 0.85°, time constant 20.48 162 ms. Scans were aggregated and the peak to peak height of the first derivative of the EPR 163 spectra was recorded as the intensity value at a given time point. Samples were taken at 164 approximately 10 min intervals across the assay time using an autosampler (Bruker 165 Corporation, MA, USA) and the running order was randomised. 166 167

Analysis of bulk fermentation volatiles in rice beers by Head Space Gas
168 chromatography (GC-HS-FID) 169 Volatile analysis was conducted using a modified version of EBC Method 9.39. Beer samples 170 were chilled to 4°C and sonicated for about 10 seconds. Degassed beer sample (10 mL) was 171 transferred to a headspace vial, 50 µl of internal standard (10,000 ppm 1-butanol) added, 172 followed by 3.5g of sodium chloride and the vial was quickly sealed tight using a crimper. for an initial 85°C for 10 min, 110°C for 13 min (ramp @ 25°C/min), 200°C for 13.25 min (ramp 179 @ 8°C/min). The temperatures of the injection port and FID detector were 150°C and 250°C, 180 respectively. 181 182

184
VDK analysis was conducted using a method based on EBC 9.42.2 which can detect and 185 quantify diacetyl (0-0.25 ppm) and 2,3-pentanedione (0-0.25 ppm) using 2,3-hexanedione as 186 an internal standard. Samples were chilled to 4°C, degassed in a cooled shaking incubator at 187 175 rpm for 5 min and filtered through a 0.45 μm syringe filter. Beer samples (5 mL) were 188 transferred to individual headspace vials, 50 µl of internal standard (5 ppm) added, followed 189 by 3.5g of ammonium sulphate and the vial was quickly sealed tight using a crimper. pressure and with helium as carrier gas. Injection volume was 500 µL at a split ratio of 1: 5. 193 Run cycle time was 12 min with an additional 20 min agitation time. The GC oven profile 194 started with an initial hold at 30°C for 2 min followed with a linear ramp to a final temperature 195 of 120°C which was held for 2 min (ramp at 70°C/min). The temperatures of the injection port 196 and ECD detector were 110°C and 150°C, respectively.

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Elemental analysis of rice beers using Inductively Coupled Plasma Mass 199 Spectrometry (ICP-MS) 200 Beer samples were degassed by sonication (5 min). All samples were diluted (1:10) with nitric 201 acid (2%) by pipetting the sample (1 mL) and nitric acid (2%, 9 mL) into a 10 mL plastic sample 202 tube. Sample tubes were capped and inverted three times. Diluted samples were stored at 203 2˚C pending elemental analysis.

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Paddy rice samples were germinated at 30°C, kilned at 70°C and hand deculmed. Both 216 varieties of rice germinated at a similar rate and after two days distinct coleoptiles were 217 visible (Figure 3). Total malting time was reduced from the 8 days 19 used in a prior study, to 218 just 3 days. This short malting time was achieved at a higher germination temperature of 30°C, 219 and would most likely result in lower malting losses (in roots and coleoptile). Under similar 220 conditions: steeping for 24 hours and germinating at 30°C, six white Thai rice cultivars have 221 been reported to result it average malting losses of 10%, 20% and 40% for a total malting time 222 of 4, 5 and 6 days respectively 10 .

224
Rice malts were mashed using the schedule shown in Figure 2. Lautering proceeded without 225 difficulty, probably due to the low gravity of the worts (ca. 9°P) and to the rice husk forming 226 an efficient filter bed. For both the rice varieties, fermentation of the wort resulted in a similar 227 decrease in pH, and specific gravity (Figure 4). This suggests that the two varieties of rice 228 produced worts of similar quality which had comparable fermentabilities (69.5 and 67.3% for 229 black and white rice worts respectively; for traditional brewing feedstocks such as barley (10% 21 ), the husk has been reported to assist 236 in an efficient lautering process 19 . However, rice husk is tougher than barley husk and of the 237 two rice varieties used, black rice has a tougher husk than the white rice husk. This could 238 create challenges in scaling up this process, especially when pumping the mash. Careful 239 milling would be required in order to balance husk preservation (to aid lautering) against 240 potential impacts on the physical properties of the mash.

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The average Free Amino Nitrogen (FAN) contents of rice worts were 83.5 and 137 mg/l for 243 black and white rice respectively (Table 2). Protein contents of the two rice varieties have 244 been previously reported to be 6.6-7.7% for Chakhao Poireiton 8,22 and 7.6-8.8% for 245 Centauro 19 . In addition to having a higher FAN content, the white rice FAN was more 246 completely assimilated by the yeast, with 89% consumption across fermentation compared 247 to 66% of FAN utilisation for the black rice beer (Table 2). However, this apparently did not 248 impact on alcohol production. The FAN content of all rice malt wort (12°P) was previously 249 reported to range between 160-179 mg/L 9 . It can be concluded that, despite the relatively 250 low wort FAN level in the present experiments, there was sufficient yeast growth to ferment 251 the wort and produce alcohol. It is likely that any brewing process based on 100% black rice 252 malt would require nitrogen-supplementation for optimal yeast health and fermentation 253 progression, particularly when brewing at higher gravities where higher wort FAN levels are 254 required 23 .

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Rice beers poured with a generous, coarse white foam and were visually somewhat hazy 257 ( Figure 5), with the white rice beer being substantially more turbid. The reasons for the 258 different turbidities in finished beer is not clear based on present data, however, one 259 possibility is that the elevated polyphenol content of black rice aided the precipitation of haze 260 active protein from the black rice beer either in the brewhouse or through 261 fermentation/maturation. It is likewise possible that the difference results from different 262 protein or starch solubilisation during processing of the 2 different varieties. The most striking 263 difference between the two beers was in terms of colour ( Figure 5, Table 2). Black rice beer 264 had a pink-orange hue (elevated a* colour co-ordinate; Phenolic content of rice beers 275 Anthocyanins were only detected in the black rice wort and beer ( anthocyanins.

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A decrease in TPC was observed between wort and finished rice beers (Table 3). This could 291 reflect losses through adsorption to yeast cells or through chill haze formation and removal. 292 For both the wort and beer samples, brewing with black rice resulted in a 4-fold greater TPC 293 than with white rice (Table 3). In lager beers the typical TPC is in the range of 150-340mg/l 25 .

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HPLC analysis indicated that both rice beers contained a broad range of phenolic compounds 295 Table 3). It was notable that protocatechuic acid was only detected in black rice worts. This 296 compound could be associated with the degradation of cyanidin-3-glucoside (or related 297 anthocyanins) as has previously been reported during cooking of black rice (Oryza sativa L. 298 japonica var. SBR) 24 . Tyrosol and indole-3-acetic acid were only detected in rice beers and not 299 in the respective worts, indicating that they were formed or imparted to the beer through 300 fermentation. There was a corresponding increase in the sum of individual phenolic acid 301 contents after fermentation: 12.06 to 25.6mg/l (black rice wort to beer) and 2.62 to 30.25 302 mg/l (white rice wort to beer). Tyrosol is an antioxidant and the Ehrlich pathway degradation 303 product of the amino acid tyrosine. Hence the greater amounts noted in white rice beers 304 (Table 3) likely corresponds with the noted higher wort FAN content (Table 2). 305 306 307 308

Flavour properties of rice beers 309
Fermentation volatiles were analysed by gas chromatography using a headspace injection 310 technique (Table 4). Concentrations of volatile esters such as ethyl acetate, ethyl hexanoate 311 and isoamyl acetate, in the rice beers were comparable with, but towards the lower end of 312 the ranges typically reported for barley malt beers (Table 4). VDK analysis indicated that the 313 black rice beer contained diacetyl (0.34 mg/L; Table 4) in excess of its flavour threshold (0.1-314 0.15 mg/L). This would be regarded as a flavour defect in conventional lager beers and was 315 most likely caused here by the noted low FAN values in black rice wort (Table 2). VDKs are 316 released into the fermenting wort and are subsequently assimilated by yeast towards the end 317 of fermentation 26 . Diacetyl is formed as an off-shoot of the pathway for valine synthesis in 318 yeast and the higher values observed in black rice worts and beer (Figure 7 and Table 4) 319 reflect: i) increased activity through the valine synthesis pathway and ii) slower uptake and 320 assimilation of diacetyl by the reduced cell mass of yeast resulting from the low wort FAN 321 content (although this did not materially impact on fermentation progression relative to the 322 white rice beer fermentations). At low concentrations diacetyl provides a butterscotch-like 323 aroma whereas pentanedione is detected as honey-like 27 . Of the two main VDK in beer, 324 diacetyl is generally present in concentrations that are approximately 10 times higher than 325 those for 2,3-pentanedione 28 . The latter was also true of the beers in this study.

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Beer foam is one of the important visual attributes by which consumers judge beer quality 29 . 328 This characteristic is influenced by both the raw materials used and the brewing process. Black 329 rice beers (257±23.8sec) had a significantly higher Nibem foam stability (FCT30) than white 330 rice beers (209±15.7sec) in this study.

332
Although detailed sensory characterization of the 100% rice beers was beyond the scope of 333 the present study, the beers were tasted by experienced brewers within our team. VDK 334 character was picked up, particularly on the black rice beers. As discussed above this could 335 readily be addressed by nitrogen supplementation of the wort prior to fermentation. 336 Furthermore, rice beers had their own individual characteristics, being slightly sour (due to 337 the lower beer pH) and with an aroma note present which was reminiscent of cooked rice 338 pudding.

340
Oxidative stability of rice beers 341 Lag time values determined using the ESR forced ageing method indicate the endogenous 342 anti-oxidative potential of a beer and are directly related to its oxidative stability. 343 Furthermore, the T150 value (signal after 150 min of the assay with PBN spin trap) is 344 commonly cited as a comparative index of the extent of radical formation after a fixed time 345 of forcing. It was immediately notable that both rice beers were unusually stable in terms of 346 their ESR forced ageing responses (Figures 6A and 6B). The traditional inflection in signal 347 intensity associated with exhaustion of the antioxidant capacity was very hard to discern for 348 100% rice beers as the ESR signals generated were relatively flat with only a gradual increase 349 in signal intensity over 150 min at 60°C. The ESR traces observed for black and white rice beers 350 were very similar to one another both in the freshly fermented and matured beers. A typical 351 ESR lag-time curve for a barley malt-derived commercial lager beer, which was run under the 352 same conditions as a part of the same experiment, is plotted (orange curve) on Figures 6A  353 and 6B by way of comparison. The question arises -were the 100% rice beers highly 354 oxidatively stable because they contained a relatively powerful array of antioxidants, or 355 because they lacked pro-oxidant species which are normally present in barley malt beers? We 356 decided to investigate this further by performing ESR forcing tests on samples generated by 357 blending proportions of each rice beer (25, 50%) into the commercial lager beer ( Figure 6C, 358 black rice; Figure 6D, white rice). Interestingly, the blending of black rice and commercial lager 359 beers resulted in the expected reductions in T150 values, in proportions that approximately 360 corresponded with the blend ratio and the T150's of the individual samples ( Figure 6C). At 361 50:50 the commercial lager/ black rice mix had a T150 value (48,600) that was around 53% of 362 that of the commercial lager beer (90,400). However, with the white rice beer, incorporation 363 at 25% made no difference to the measured T150 relative to the commercial lager beer and 364 even in a 50:50 blend ratio the T150 value (72,900) was as much as 81% of that in the 365 commercial lager alone. Based on these results it is speculated that 100% rice beers lack 366 significant pro-oxidant species which are present in malt derived lager beers and also that 367 they contain antioxidant species which can enhance the antioxidant capacity of beers. 368 Furthermore, it can be concluded that the black rice beers contained species which improved 369 the oxidative stability of the commercial lager beer when the two were mixed in blends and 370 that this trend was more evident when blending in the black rice beer as opposed to the white 371 rice beer (comparing Figures 6 C & D). 372 373

Spectrometry (ICP-MS)
375 Thirty-one metallic elements, including almost all essential and toxic metals such as lead, 376 cadmium, mercury, arsenic, silver, and thallium, were quantified in both of the beers by ICP-377 MS (Table 5). For comparative purposes a 'control lager' beer brewed from 100% barley malt 378 was submitted for analysis alongside the rice beers to highlight major differences in elemental 379 composition relative to the primary grist materials used.

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Rice beers were relatively rich in magnesium and contained less potassium than the control 382 lager. When considering the oxidative stability of beers there is much focus on the 383 concentrations of transition metals such as iron, copper and manganese, which can catalyse 384 the formation of pro-oxidant radical oxygen species 18 . In view of the noted oxidative stability 385 of 100% rice beers it is interesting to note that they contained very low levels of iron and 386 copper relative to the control lager (Table 5). However, the converse was true of manganese 387 which was present at mg/L quantities, more than 10-fold higher than in the control lager 388 (177.8 μg/L). Apparently this did not damage the oxidative stability of the 100% rice beers, 389 perhaps due to the form in which manganese is present when brewing with 100% rice. This 390 may favour the extraction of manganese through the brewing process, since reports 391 elsewhere in the scientific literature of typical manganese contents of the raw materials 392 themselves do not suggest such a high discrepancy as was noted here in the finished beers 393 (raw rice 21 mg/kg dry weight, raw wheat 31 mg/kg and raw barley 29 mg/kg 30,31 ).

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Arsenic concentrations in rice beers (14.86 µg/l and 27.9 µg/l for black and white rice beer 396 respectively) are of particular interest due to concerns about arsenic contents in rice. These 397 results will reflect differences in the mineral contents due to the soil of the cultivation areas 398 (India vs Italy) and also the cultivars (black vs white). Arsenic content of Italian beers has been 399 reported to be 3-24 µg/l 32 , Polish beer ranged from 2-13 µg/l 33 and for beers bought in New 400 York   Data are the mean of two biological replicates ± SD; ND = not detected.