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Further Development of the Stevia Natural Sweetener Industry

AUGUST 2012 RIRDC Publication No. 12/051

Further Development of the Stevia Natural Sweetener Industry by David J Midmore, Andrew Rank, Kerry B Walsh, Ria Reyes, Geeta Gautam and Kylie C Hopkins

August 2012 RIRDC Publication No. 12/051 RIRDC Project No. PRJ-002426

© 2012 Rural Industries Research and Development Corporation. All rights reserved.

ISBN 978-1-74254-394-9 ISSN 1440-6845 Further Development of the Stevia Natural Sweetener Industry Publication No. 12/051 Project No. PRJ-002426 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165. Researcher Contact Details Professor David J Midmore Centre for Plant and Water Science, Central Queensland University, Rockhampton, Qld 4702.

Email: [email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Alan Davey, Senior Research Manager - New Plant Industries Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604

Phone: Fax: Email: Web:

02 6271 4100 02 6271 4199 [email protected]. http://www.rirdc.gov.au

Electronically published by RIRDC in August 2012 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313

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Foreword The research conducted within this project, jointly funded by RIRDC, Central Queensland University (CQU) and Sanitarium, is of great importance for those who consume sweetener alternatives to sugars, whether for health or weight management reasons. Having locally sourced and processed natural alternatives to manufactured sweeteners is important to the Australian consumer and the broader food industry. This report shows advances in the field trial and laboratory analyses of steviol glycosides produced from plant material. A non-invasive tool for discriminating between large scale differences in steviol glycoside concentrations between different genotypes in the field is described. Studies show how flowering times can be manipulated to influence steviol glycoside production, and the success in mass selecting for later flowering. The report also documents visual symptoms and describes nutrient and soil requirements for Stevia. There is also a set of experiments described which identify a suitable herbicide for weed control in Stevia. This report makes a substantial contribution to the establishment of a stevia industry in Australia. There will always be a need for an ongoing research program to support and strengthen the industry, especially if it is to grow and maintain a sizable share of the world market. Although, it will be some years before the industry will be established in Australia, it is gaining much ground in the USA, with similar climatic conditions to Australia; hence it is important that Australia not trail after making the headway in having steviol glycosides approved as intense sweeteners in a range of food products. The literature does not indicate any proneness to weediness in Stevia rebaudiana. However, as with the introduction of any species into a new environment, RIRDC recommends potential growers seek advice from local agronomists before planting. This report is an addition to RIRDC’s diverse range of over 2000 research publications and it forms part of our New and Developing Plant Industries R&D program, which aims to facilitate the development of new industries based on plants or plant products that have commercial potential for Australia. Most of RIRDC’s publications are available for viewing, free downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.

Craig Burns Managing Director Rural Industries Research and Development Corporation

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About the Authors Professor David Midmore is the Foundation Professor of Plant Sciences at CQUniversity and Director of the Centre for Plant and Water Science, positions held since 1995. He has extensive experience in tropical agriculture and development of new industries, in Australia and overseas. With Mr. Rank he has previously researched aspects of Stevia rebaudiana, funded by RIRDC. Mr Andrew Rank is a Research Fellow with the Centre for Plant and Water Science and has been an agricultural consultant for over 30 years in temperate and tropical regions as well as having experience with regional and industry level planning, development, research and economic projects in Australia and overseas. Professor Kerry Walsh is Professor of Plant Sciences at CQUniversity and has nearly two decades research experience with the use of NIR in non-invasive contexts. He has commercialised the use of NIR in the improvement of fruit quality. Dr Ria de Guzman (nee Reyes), originally from the Philippines, completed her PhD on stevia with CQUniversity in 2010 and currently works for Langdon Ingredients. Ms Geeta Gautam, from Nepal, completed her MAppSci with CQUniversity on stevia in 2011. Ms Kylie Hopkins is a research technician with the Centre for Plant and Water Science.

Acknowledgments We thank: RIRDC and Sanitarium Plc for their financial contribution to the activities presented in this report, Dr John Ashton, Strategic Research Manager, Sanitarium Development & Innovation for helpful insights into the sweetener industry and for donating Soolite (high rebaudioside A sweetener) for use in the analysis. Phazir Polychromix (http://www.polychromix.com/) Polychromix Wilmington MA 01887 USA for analyses of some leaf samples, Craig Hall of AustChilli, Bundaberg and Deb and Alan Power of “Nenagh", Yeppoon for generous loan of land for trials with herbicides, Linda Ahern for her support in formatting this report. We gratefully acknowledge the following for freely supplying herbicide samples: Nufarm, Dupont, MacScpread Australia, Agnova, Syngenta, Agricrop and Bayer. We also thank the many other contacts in the industry for their generous exchange of information.

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Contents Foreword ............................................................................................................................................... iii About the Authors ................................................................................................................................ iv Acknowledgments................................................................................................................................. iv Executive Summary ............................................................................................................................ xii Introduction ........................................................................................................................................... 1 Objectives ............................................................................................................................................... 4 Methodology .......................................................................................................................................... 5 1. a) Further refinement of procedures for the reliable and accurate analysis of all glycosides using HPLC. .................................................................................................................... 5 1. b) Further development of technology based on near infrared spectroscopy (NIRs) scanning for rapid assay of glycoside content in plant material for use in variety selection work and for grading (on-farm or at processing mill receival) of dried stevia hay to determine quality grades as the basis for the payment to growers. .................................................. 7 2. Plant physiological and basic management practices that influence or control flowering, glycoside accumulation, ratoon performance and total yield while optimising crop returns. .................................................................................................................................... 10 3. Evaluation of weed control practices and herbicides to provide data for registration or permit-use of suitable chemicals to use with stevia. .................................................................. 17 4. Providing support for a limited number of farmers growing trial areas of stevia. This support includes provision of planting material and leaf analysis of plot samples. ...................... 20 Results .................................................................................................................................................. 21 1. a) To further refine the procedures for the reliable and accurate analysis of all glycosides using HPLC. .................................................................................................................. 21 1. b) To further develop a technology based on near infrared spectroscopy (NIRs) scanning for the rapid assay of glycoside content in plant material for use in variety selection work and for grading (on-farm or at processing mill receival) of dried stevia hay to determine quality grades as the basis for the payment to growers. ............................................ 28 2. To undertake studies on plant physiology and basic management practice to determine factors (including stresses) that influence or control flowering, glycoside accumulation, ratoon performance and total yield, thereby to optimise crop returns. ................... 44 3. To evaluate weed control practices and herbicides to provide data for registration or permit-use of suitable chemicals to use with stevia........................................................................ 73 4. To provide support for a limited number of farmers growing trial areas of stevia. This support includes provision of planting material and leaf analysis of plot samples. .............. 76

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Further development of analysis methods, especially NIR scanning for rapid assay..................... 77 Basic physiology and agronomy studies as a foundation for future variety selection and crop management practices ............................................................................................................. 82 Evaluation of weed control practices and herbicides to provide data for approval of chemical use by permit or registration............................................................................................ 93 Implications.......................................................................................................................................... 96 Recommendations ............................................................................................................................... 96 References and Publications............................................................................................................... 97

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Tables Table 1

Day length (h) not including civil twilight for whole months at weekly intervals at Rockhampton (23° 23' S). ........................................................................................................................................ 11

Table 2

Day length (h) associated with growth stages in stevia experiments in the open at Rockhampton. .. 11

Table 3

Average monthly maximum and minimum air temperatures (°C) during experiments in the open at Rockhampton. ...................................................................................................................... 11

Table 4

pH and the amount of buffer added to the total volume of half-strength hydroponic solution (Manutec). ........................................................................................................................................ 17

Table 5

Herbicide and other treatments for the Yeppoon field trial. ............................................................. 18

Table 6

Herbicide application conditions for Bundaberg field trial. ............................................................. 19

Table 7

Slope and correlation coefficient of linear regression of peak area to stevioside concentration, in relation to the range of stevioside concentrations used in the calibration set (data of Figure 5)... 24

Table 8

Comparison of HPLC peak areas of filtered and unfiltered stevioside standards. XC cartridge was used to filter the standards. ........................................................................................................ 26

Table 9

PLSR calibration statistics for models of (A) rebaudioside A and (B-E) Soolite in solution, based on different wavelength ranges (using an Antaris FTNIR instrument). .................................. 34

Table 10

Population statistics (sample size, mean and standard deviation) of total SG, stevioside and rebaudioside A content in leaf material (% w/w) of populations used for the calibration and validation of models for each NIR instrument. ................................................................................. 36

Table 11

Cross validation statistics (correlation coefficient, RCV, and root mean square error of cross validation, RMSECV) for a PLS regression models based on total SG as determined by HPLC and four wavelength ranges of d2(log 1/R) spectra collected with three instruments, and using the same leaf material in three formats. ............................................................................................ 37

Table 12

Calibration statistics (correlation coefficient, RCV and root mean square error of cross validation, RMSECV) for models of stevioside and rebaudioside A (% w/dw) of the d2(log 1/R) spectra of dry ground stevia leaf. ...................................................................................................................... 38

Table 13

Calibration statistics (correlation coefficient, RCV and root mean square error of cross validation, RMSECV) for models of total SG (% w/dw) of spectra of dry ground stevia leaf, using different spectral pre-processing methods.. ..................................................................................................... 38

Table 14

Summary of correlation (R) and root-mean-square-error-of-prediction (RMSEP) for the prediction of total SG in two wavelength windows, and stevioside and rebaudioside A leaf concentration (% dw) in one wavelength window, of 55 independent leaf samples. ........................ 41

Table 15

Population statistics of the first and second half of the combined calibration and prediction set for the NIRS 6500 and FTNIR Nicolet Antaris unit. ........................................................................ 42

Table 16

Prediction statistics for total SG, stevioside and rebaudioside A using a prediction set of unique spectra, but of the same population that the calibration set was drawn from. ................................... 42

Table 17

Calibration statistics (correlation coefficient, R, and root-mean-square-error-of-prediction, RMSEP) for models based on subset samples from the original calibration set (Model 1), the original prediction set (Model 2) and their combination (Model 3). ................................................ 43

Table 18

Days to flower and duration of flowering for three stevia varieties transplanted on four dates in the seed-established experiment (n=50)............................................................................................ 44

Table 19

Average number of days from transplant to flower of three stevia varieties transplanted on four dates in the seed-established experiment (n=15). ............................................................................. 45

Table 20

Days to first flower and 50% flowering of three stevia varieties as affected by days after ratooning (DAR) (n=50). .................................................................................................................. 45

Table 21

Average numbers of days to flower expressed as affected by the number of days after ratooning of three stevia varieties. .................................................................................................................... 46

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Table 22

Effect of seedling age on above ground biomass (dry weight, g/plant) at 12 weeks from transplant of three stevia varieties in the seed-established experiment. ............................................ 46

Table 23

Effect of seedling age on plant height (cm) at 12 weeks from transplant of three stevia varieties in the seed-established experiment. .................................................................................................. 47

Table 24

Effect of seedling age on leaf yield (dry weight, g/plant) at 12 weeks from transplant of three stevia varieties in the seed-established experiment. .......................................................................... 47

Table 25

Effect of seedling age on stem yield (dry weight, g/plant) at 12 weeks from transplant of three stevia varieties in the seed-established experiment. .......................................................................... 47

Table 26

Average biomass yield/plant (g) of three varieties of stevia in the ratoon crop. ............................... 48

Table 27

Average leaf dry weight/plant (g) of three varieties in the ratoon crop. ........................................... 48

Table 28

Average stem dry weight/plant (g) of three varieties in the ratoon crop as affected by days after ratooning. .......................................................................................................................................... 48

Table 29

Average plant height (cm) of three varieties of stevia in the ratoon crop. ........................................ 49

Table 30

Average total stevioside concentration (% dry weight) in leaves of three varieties of stevia in the seed-established experiment. ...................................................................................................... 49

Table 31

Average rebaudioside A concentration (% dry weight) in leaves of three varieties of stevia in the seed-established experiment. ...................................................................................................... 50

Table 32

Average total SG concentration and content in leaves (mg/plant) of three varieties of stevia grown at different transplanting ages in the seed-established experiment. ....................................... 50

Table 33

Flowering of F1 plants for stevia variety selections for early, medium or late flowering (days after sowing). .................................................................................................................................... 51

Table 34

Average plant height (cm) in early, medium and late populations from stevia varieties in their F1 generation.. .................................................................................................................................. 51

Table 35

Average above ground biomass (g/plant) in early, medium and late populations from stevia varieties in their F1 generation. ........................................................................................................ 51

Table 36

Average leaf dry weight (g/plant) in early, medium and late populations from stevia varieties in their F1 generation. ........................................................................................................................... 51

Table 37

Flowering of stevia varieties and flowering groups under 24 hour photoperiod............................... 52

Table 38

Average plant height (cm) of stevia varieties and flowering groups under 24 hour photoperiod. ... 52

Table 39

Average above-ground biomass (g/plant) of stevia varieties and flowering groups under 24 hour photoperiod....................................................................................................................................... 53

Table 40

Average leaf dry weight (g/plant) of stevia varieties and flowering groups under 24 hour photoperiod....................................................................................................................................... 53

Table 41

Average stem dry weight (g/plant) of stevia varieties and flowering groups under 24 hour photoperiod....................................................................................................................................... 53

Table 42

Flowering of stevia varieties and flowering groups under 16 hour photoperiod............................... 54

Table 43

Average plant height (cm) of stevia varieties and flowering groups under 16 hour photoperiod. .... 54

Table 44

Average stem dry weight (g/plant) of stevia varieties and flowering groups under 16 hour photoperiod....................................................................................................................................... 55

Table 45

Flowering of stevia varieties and flowering groups under 14 hour photoperiod............................... 55

Table 46

Average plant height (cm) of stevia varieties and flowering groups under 14 hour photoperiod. .... 56

Table 47

Average above-ground biomass (g/plant) of stevia varieties and flowering groups under 14 hour photoperiod....................................................................................................................................... 56

Table 48

Average leaf dry weight (g/plant) of stevia varieties and flowering groups under 14 hour photoperiod....................................................................................................................................... 57

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Table 49

Average stem dry weight (g/plant) of stevia varieties and flowering groups under 14 hour photoperiod....................................................................................................................................... 57

Table 50

Days to first flowering and percent of flowering at the time of harvest (6 weeks) of stevia varieties and flowering groups under 12 hour photoperiod. ............................................................. 57

Table 51

Average number of days to flower of three stevia varieties and flowering groups under 12 hour photoperiod....................................................................................................................................... 58

Table 52

Average plant height (cm) of stevia varieties and flowering groups under 12 hour photoperiod. .... 58

Table 53

Average above-ground biomass (g/plant) of stevia varieties and flowering groups under 12 hour photoperiod....................................................................................................................................... 59

Table 54

Average leaf dry weight (g/plant) of stevia varieties and flowering groups under 12 hour photoperiod....................................................................................................................................... 59

Table 55

Average stem dry weight (g/plant) of stevia varieties and flowering groups under 12 hour photoperiod....................................................................................................................................... 59

Table 56

Mean height and dry weight of stem, leaf, root and total above ground biomass (g/plant) and leaf to stem ratio at harvest as affected by soil moisture................................................................... 60

Table 57

Mean stem water potential and osmolality as affected by soil moisture. .......................................... 60

Table 58

Effect of soil moisture on photosynthetic rate, transpiration rate, stomatal conductance and chlorophyll content. .......................................................................................................................... 61

Table 59

Percent dry weight of stevioside and rebaudioside A in leaves of stevia and their sum (total SG) and total SG per plant grown at different soil moisture content. ...................................................... 61

Table 60

Average SPAD readings for stevia leaves on plants grown in various nutrient solutions. ................ 69

Table 61

Photosynthetic rate, transpiration rate and stomatal conductance of stevia at the time of harvest (at four weeks of treatment application).. ......................................................................................... 70

Table 62

Effect of different nutrient deficiencies on stevioside and rebaudioside A concentration and content. ............................................................................................................................................. 71

Table 63

Dry weight of stem, leaf, root and total biomass, shoot to root ratio, plant height and number of branches per plant grown in solution at different pH. ....................................................................... 72

Table 64

Photosynthetic rate, transpiration rate, stomatal conductance and estimated chlorophyll concentration (SPAD) of stevia grown in solution at different pH. .................................................. 72

Table 65

Percent dry weight of stevioside and rebaudioside A in leaves of stevia grown at different pH. ..... 73

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Figures Figure 1

Typical chromatogram of leaf water extract of dry ground stevia leaves with stevioside and rebaudioside A retention time of 9.504 and 13.643 min respectively. ........................................... 6

Figure 2

Estimated leaf stevioside content (% dw basis) for extraction using two types of leaf formats: dry (___) and fresh (- - -) ground leaves and two types of extraction solvent: water () and 70% ethanol () in a single 10 mL extraction protocol.. ................................................................................... 21

Figure 3

HPLC chromatogram of 1 g/L stevioside in (A) 70% ethanol (peak area = 2695) and (B) water (peak area = 2403) and 1 g/L rebaudioside A in (C) 70% ethanol (peak area = 2089) and (D) water (peak area = 2339). ........................................................................................................................ 22

Figure 4

Peak area plotted against amount of (A) stevioside and (B) rebaudioside A delivered in two injection volumes: 5 µL (◊) and 10 µL (). Samples were water based. ...................................... 23

Figure 5

Correlation coefficient of linear regression of peak area to stevioside concentration, with respect to maximum concentration of stevioside in the calibration set.. ........................................................ 24

Figure 6

Change in retention time of rebaudioside A with mobile phase composition. ............................... 25

Figure 7

HPLC peak area (,) and peak height (____ , _ __ _) of stevioside standards (10 µL injection of 10, 7.5, 5, 2.5, 1, 0.5, 0.25 and 0.125 g/L in 70% EtOH) that were unfiltered (shaded symbol) and filtered (open symbol), respectively with an XC cartridge. ..................................................... 26

Figure 8

Final clean-up protocol for SG standards and leaf extracts. A pre-conditioning step for the NH2 cartridge was included. .................................................................................................................. 27

Figure 9

Peak area of ethanol-based standards (0.125, 0.25, 0.5 and 1 g/L) of stevioside (filtered  and unfiltered ____) and rebaudioside A (unfiltered __◊__ and filtered ) using the final SPE clean-up protocol. ......................................................................................................................................... 28

Figure 10

NIRs spectra of powdered crystalline samples of stevioside (—), rebaudioside A (___), sucrose (…..), starch (_._._) and salt (_ _ _) using a transmission optical geometry (Antaris FTNIR unit). Arrows point to major features of the SGs. ................................................................................................ 29

Figure 11

Raw absorbance spectra (A), difference spectra (to water) (B), specific absorbance spectra (C) and d2A spectra (D) of rebaudioside A (Soolite) standards (0.01% (—), 0.02% (_ _ _), 0.04% (_._._) and 0.06% (…..)) scanned at a fixed temperature of 40 °C. .......................................................... 30

Figure 12

Absorbance spectra (around 1400 nm) of water at (A) 23 °C (—), 29 °C (_ _ _), 40 °C (_ . _) and 50 °C (…..), and (B) at 40 °C, but in the presence of sucrose at 1% (_ _ _), 10% (_ . _ . _) and 20% w/v (…..). ............................................................................................................................................... 32

Figure 13

Absorbance spectra (around 2000 nm) of water at 40 °C with sucrose content of 1% (_ _ _), 10% (_ . _ . _) and 20% (…..). ............................................................................................................... 32

Figure 14

Calibration model statistics for PLS regression models of Soolite in aqueous solutions, based on various wavelength regions (start wavelength displayed on the x axis, end wavelengths on the y axis) – (A) Number of PLS factors, (B) RMSEC, and (C) RMSECV. .......................................... 33

Figure 15

NIR reflectance spectra (log 1/R) of stevia leaves using an FTNIR Nicolet Antaris unit. ............. 35

Figure 16

NIRs reflectance spectra of fresh (—), dry (_ _ _) and dry ground (……) stevia leaves using the NIRS 6500 unit. Arrow indicates chlorophyll absorption peak. .............................................................. 35

Figure 17

PLS regression coefficients for the model based on NIR6500 spectra (1100-2500 nm region) of dry ground stevia leaves. ...................................................................................................................... 39

Figure 18

Comparison of different wavelength regions used in PLS regression models on total SG content, based on d2(log1/R) spectra of dried ground leaf, collected with the NIR6500 instrument. . ...... 40

Figure 19

Total above ground biomass (a) and leaf yield (b) of three stevia varieties across three flowering groups under 16 hours photoperiod. Lsd refers to the interaction between flowering groups and varieties.......................................................................................................................................... 55

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Figure 20

Nutrient deficiency symptoms in stevia; a) NPK, b) N, c) P, d) K, e) Ca, f) Mg, g) S, h) B, i) Micro nutrient, j) Manganese, k) Cu, l) Iron, m) Mo, n) Zn, o) Cl and p) Complete. Nutrient omission was imposed on 13 week plant for four weeks...................................................................................... 63

Figure 21

Effects of nutrient deficiency in stevia roots; a) NPK, b) N, c) P, d) K, e) Ca, f) Mg, g) S, h) B, i) Micro nutrient, j) Manganese, k) Cu, l) Iron, m) Mo, n) Znand o) Cl. .......................................... 65

Figure 22

Average total above ground dry weight of stevia (Stevia rebaudiana) grown with various nutrient deficiencies. Plants harvested after 4 weeks of treatment imposition. Treatments with the same letter did not differ significantly from each other at P