Investigation of the stabilization and preservation of sweet sorghum juices

Industrial Crops and Products 64 (2015) 258–270

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Investigation of the stabilization and preservation of sweet sorghum juices Gillian Eggleston a,∗ , Anthony DeLucca a , Scottie Sklanka a , Caleb Dalley b , Eldwin St. Cyr a , Randall Powell c a

USDA-ARS Southern Regional Research Center, 1100 Robert E. Lee Blvd. , New Orleans, LA, 70124, USA USDA-ARS Sugarcane Unit Houma, LA 70360, USA c Delta BioRenewables, LLC. Memphis, TN, USA b

a r t i c l e

i n f o

Article history: Received 28 May 2014 Received in revised form 20 August 2014 Accepted 5 September 2014 Available online 11 October 2014 Keywords: Sweet sorghum juice UV irradiation Juice preservation Clarification Pasteurization Microbial inactivation

a b s t r a c t Sweet sorghum juice is extremely vulnerable to microbial spoilage during storage because of its high water activity and rich sugar medium, and this represents a major technical challenge. The effects of clarification (80 ◦ C; limed to pH 6.5; 5 ppm polyanionic flocculant) and UV-C irradiation were investigated as stabilization and preservation treatments for juices stored at ambient temperature (∼25 ◦ C). Juices were extracted by roller press from various sweet sorghum cultivars grown in humid and dry environments in Louisiana and Tennessee, respectively. Raw juices contained up to 109 total bacteria cfu/mL. Initial results indicated that pilot plant clarified juice was considerably more stable than raw or UV-C irradiated (15 W lamp aquaculture system at ∼25 ◦ C) juice, irrespective of cultivar. Further experiments were undertaken to elucidate if heating (80 ◦ C; 30 min) or impurity precipitation or both of these components of the clarification process were responsible for improved juice stability. Clarification or heating both achieved 3- to 4-log reductions of lactic acid bacteria in juices to negligible levels (<150 cfu/mL), and also significantly (P < 0.05) reduced total bacterial counts. Juice heating gave similar results as the whole clarification process up to ∼24 h storage, but became slightly worse between 24–28 h. Overall, clarified or heated (80 ◦ C; 30 min) juice stored at 25 ◦ C can be stored for at least 48 h before unacceptable spoilage occurs. Fingerprint ion chromatography with integrated pulsed amperometric detection (ICIPAD) oligosaccharide profiles can be used to monitor sweet sorghum juice spoilage >100 cfu/mL lactic acid bacteria. Published by Elsevier B.V.

1. Introduction While much research has been focused on process optimization for advanced and second generation biofuels, attention is now focused on developing sustainable supply chains of sugar feedstocks for the new, flexible biorefineries (Koninckx, 2013). This includes improved feedstock quality and cost-effective approaches for minimizing feedstock sugar losses during storage (Koninckx, 2013). Sweet sorghum (Sorghum bicolor L. Moench) is a type of sorghum that contains a high concentration of soluble sugars in the

Abbreviations: Rt , Retention time; UV-C, Irradiation Ultra Violet light at 254 nm; DNA, Deoxyribonucleic acid; Aw , Water activity; HMW, High molecular weight; MOL, Milk of lime; GRAS, Generally recognized as safe; GL, Green leaves; BL, Brown leaves; NTU, Nephalometer turbidity units; CJ, Clarified juice; IC-IPAD, Ion Chromatography with Integrated Pulsed Amperometric Detection. ∗ Corresponding author. Tel.: +1 504 286 4446; fax: +1 504 286 4367. E-mail address: [email protected] (G. Eggleston). http://dx.doi.org/10.1016/j.indcrop.2014.09.008 0926-6690/Published by Elsevier B.V.

plant sap or juice. It is an attractive biomass feedstock because of its efficient C4 photosynthetic pathway, easy cultivation from seed, low fertilizer and water requirements, wide geographical suitability, and huge breeding potential (Eggleston et al., 2013). For the large-scale, commercial manufacture of bio-based fuels and chemicals from sweet sorghum, fundamental processing questions urgently need to be addressed, especially those associated with seasonal production. Sweet sorghum juice is extremely vulnerable to microbial spoilage on storage because of its high water activity (Aw ) and rich sugar medium (Wu et al., 2010). It is also affected by enzymatic and acid deterioration reactions but to a much lesser degree than microbial deterioration (Eggleston, 2002; Singh et al., 2006). Juice microbial spoilage or deterioration represents a major technical challenge during the stabilization and storage of juices for any length of period (Wu et al., 2010). Thus, stabilization and preservation technologies for juices are needed to prevent loss of fermentable sugars and allow for the maximum fermentation yields of end-products including biofuels and bioproducts.

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Little has been reported on sweet sorghum juice stability and storage. Daeschel et al. (1981) observed that fresh sweet sorghum juice spoiled within 5 h at ambient temperatures. Wu et al. (2010) reported that at room temperature (∼25 ◦ C) up to 12–30% fermentable sugars can be lost in 3 days, and 40–50% in 1 week. Bacterial counts increased 30 to 300-fold in the first week and tended to be mostly lactic acid bacteria. Wu et al. (2010) also reported that at refrigeration temperature (4 ◦ C) sweet sorghum juices lost less than 1 and 3% total sugars after 1 and 2 weeks, respectively. However, at the commercial scale refrigeration is extremely expensive. Preservation processes for foods and beverages are typically through chemical or physical processes, and include heating, oxidation, water removal, osmotic inhibition, freezing, irradiation, and chemical methods (Rector, 2012). All methods reduce the amount of microorganisms, including bacteria, protozoa, viruses, yeasts, and fungi. Although stabilization of juices is possible with chemicals including benzoates, nitrites, sulfites which inhibit the activity of bacteria or kill the bacteria, and other biocides, they may interfere with end-product fermentation. However, a biocide that evaporates after its use would be useful. Choride dioxide (ClO2 ) has been used as a bacterial decontaminant of water and equipment, and against numerous lactic acid bacteria in alcoholic fermentations (Meneghin et al., 2008) and is known to evaporate after usage. Ultraviolet (UV) light radiation has been used for many years in pharmaceutical, electronic, aquaculture, and maple sugar industries, and more recently in food and beverage industries, to inactivate many types of microorganisms (Sipple et al., 1970; Morselli and Whalen, 1983; Guerrero-Beltran and BarbosaCanovas, 2004; Knights, 2013). Although the UV light range is from 100 to 400 nm, it is the narrow UV range of 200–280 nm (UV-C range) that is considered the germicidal range (Bolton, 1999); the highest germicidal effect is between 250 and 270 nm (Bachmann, 1975). For this reason, a wavelength of 254 nm (UV-C) generated by low-pressure mercury (LPM) lamps is used to disinfect surfaces, water, and some food and beverage products (Guerrero-Beltran and Barbosa-Canovas, 2004). In liquid foods and beverages, UV-C acts as a photocatalyst, inactivating only the DNA of the pathogen in the product. Specifically, the cellular DNA of the microorganism absorbs UV-C radiation (non-ionizing radiation) and is damaged. As a consequence, the microorganism becomes reproductively inactive and is unable to replicate and eventually leads to cell death. The amount of DNA damage is proportional to the amount of UV-C exposure (Miller et al., 1999), and can be reversed depending on the UV repair system of the microorganism. This phenomenon is reflected in the sigmoidal shape of the curve for microbial inactivation (Anon, 2013). Sufficient energy and duration is needed to achieve the lethal UV-C dosage necessary for microorganisms and disinfect the surface of the liquid (Knight, 2013). Different bacteria require difference dosage levels of UV-C to be inactivated. Relatively few literature reports are available on process factors affecting microbial inactivation by UV-C (Anon, 2013). Pressure, temperature, and pH of the medium have little effect whereas product composition, solids content, color, starches and food chemistry in general have an effect, but literature reporting these individual factors is not available (Anon, 2013). Compared to raw sugarcane juice, clarified sugarcane juice (CJ) is more stable due to removal of microorganisms, enzymes, and other suspended and turbid impurities through heating and precipitation. Our research team recently developed a clarification method for sweet sorghum juices (Andrzejewski et al., 2013a,b), but the exact role clarification plays in preservation of sweet sorghum juice against spoilage was still unknown. Eggleston et al. (2014a,b) reported that clarified sweet sorghum syrups, with many suspended solids and turbid particles removed, stored better than raw

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syrups because they are less susceptible to microbial deterioration. The degree of juice clarification also affects heat transfer in subsequent evaporators, end product yields and quality. Clarification of sweet sorghum juices includes a combination of thermal, chemical, and precipitation processes (Andrzejewski et al., 2003a,b). The juice is first heated to allow colloidal particles, particularly proteins, to coagulate and form natural flocs. Hydrated lime (calcium hydroxide) is then added in the form of milk of lime (MOL), to the heated sweet sorghum juice to increase the pH to 6.5 for (i) neutralization of acids, (ii) reduction of unwanted acid degradation of invert sugars in downstream thermal evaporation, (iii) formation of calcium phosphate flocs, and (iv) introduction of positively charged particles in the juice solution (Andrzejewski et al., 2013a,b). Calcium, from the juice and MOL, and phosphorous that occurs naturally in the sweet sorghum juice are necessary in the formation of calcium-phosphate bridges that aid in the flocculation process. Floc precipitation is further aided by HMW polyanionic acrylamide flocculants which agglomerate particles and add weight increasing settling rates. Both the lime and flocculant are GRAS. This study on the stabilization and preservation of raw juices extracted from sweet sorghum was undertaken at the request of industry. The use of biocides and other chemical preservatives was not investigated as these could possibly interfere with and reduce potential fermentation yields downstream. 2. Material and methods 2.1. Chemicals Powdered, hydrated lime (Carmeuse, Pittsburgh, PA, USA) and flocculant (Praestol 2640 z, ∼2,700,000 MW copolymer of acrylamide and sodium acrylate with a medium charge density [Stockhausen, Krefeld, Germany]) were kindly provided by the staff at Lafourche Sugars, LLC (Thibodaux, LA, USA). Flocculant (1 g) was added to 1 L of distilled water (0.1% solution) and allowed to thoroughly mix for 1 h and sit for 24 h before use. Analytical grade hydrochloric acid, sodium hydroxide, and triethanolamine were purchased from Sigma-Aldrich (St. Louis, MO). Sodium acetate trihydrate came from Fisher Scientific (Fair Lawn, NJ, USA). 2.2. Sweet sorghum production and juice extraction 2.2.1. Production of sweet sorghum juice in Louisiana Two commercial sweet sorghum cultivars (Theis and Top 766 aka Topper) were planted on May 10, 2013 at the Ardoyne Farm of the USDA-ARS Sugarcane Research Unit in Schriever, LA. Plots consisted of a single raised bed row measuring 1.8 m wide and were at least 107 m in length with four replicates. The soil type was Schriever clay, and plots were fertilized as normal for sorghum (90 kg/ha N, 22 kg/ha K, 45 kg/ha P) and treated with metolachlor plus atrazine (1.42 plus 1.2 kg/ha) at planting followed by pendimethalin 1.6 kg/ha) upon reaching the four leaf growth stage. On Sept 10, 2013, multiple whole-stalks of mature Theis were harvested by hand and topped. The top quarter of the stalks had green leaves (GL) and the rest of the stalk had brown leaves (BL); some secondary or auxiliary seed heads were visible. The average weight per bundle was 15.6 ± 1.6 kg and the average weight per stalk was 0.8 ± 0.1 kg. Juice was extracted, from 50 bundles of 20 whole-stalks, through a SquierTM (Buffalo, NY, USA) three-roller mills at Ardoyne Farm (Houma, LA). First and second expressed juices were collected and combined and no imbibition water was added. The green colored juice was filtered through a 0. 6 mm mesh filter. Raw juice was immediately transported (∼1 h) in drums placed in a cargo tote (1.2 × 1.0 × 1.2 m) containing a rock salt (sodium chloride)-water ice bath (6 ◦ C) to the USDA-ARS-Southern

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Regional Research Center (SRRC) pilot plant in New Orleans where a portion of the juice was clarified, as described in Section 2.3. The limed juice target pH was 6.5 (Andrzejewski et al., 2013a,b). On Sept 17, 2013, fifty bundles (twenty whole-stalks per bundle) of mature Top 76-6 were harvested by hand and topped, and juice was similarly extracted, transported, and processed into raw and clarified juice similar to the Sept 10 samples. The top one eighth of the Top 76-6 stalks had GL as and the rest of the stalk had BL; some secondary or auxiliary seed heads were visible. The average weight per bundle of Top 76-6 was 16.1 ± 1.7 kg and the average weight per stalk was 0.8 ± 0.1 kg. The raw Top 76-6 juice was brown in color. The raw and clarified juices (CJs) from Theis and Top 76-6 were subsequently subjected to UV light and storage experiments as described in Section 2.5. 2.2.2. Production of sweet sorghum juice in Tennessee In 2013, sweet sorghum juice was provided by Delta BioRenewables, Memphis, TN. Specifically, juice was obtained from sweet sorghum commercial cultivar M81E on Sept 4, 2013 after planting on April 18, 2013, Top 76-6 on Oct 22, 2013 (planted on May 5), Top 76-6 on Nov 5, 2013 (planted on May 5) and KN Norris on Nov 19, 2013 (planted on May 30). Sweet sorghum billets (∼25 cm length) were harvested with a Case New Holland (Burr Ridge, IL) sugarcane combine harvester. Seed heads were removed by the sugarcane harvester topper. The sweet sorghum billets were crushed by passing through a custom built 4-roll mill (Jeffersonville, KY, USA) followed by a Laurel Machine and Foundry Co (Laurel, MS, USA) 3-roll mill. The combined first and second expressed juices were mixed and filtered through a 0.6 mm pore size screen and stored in three 114 L drums. The juice was transported in a cargo tote (1.2 × 1.0 × 1.2 m) containing a rock salt-water ice bath, with ice and salt added as necessary to maintain a temperature below 6 ◦ C during transport (∼6 h) to the USDA-ARS pilot plant in New Orleans, LA. At the USDA pilot plant, CJs were produced as described in Section 2.3. The target limed pH for clarification was ∼6.5 for all four sample dates (Andrzejewski et al., 2013a,b). The raw and CJs were then subjected to UV treatments and storage experiments as described in Section 2.5. On Nov 5 and 17 heated juices were also produced as described in Section 2.4, and subjected to storage experiments. 2.3. Pilot plant manufacture of clarified juice (CJ) CJ was produced from sweet sorghum raw juice in the USDAARS-SRRC pilot plant (for details see Eggleston et al., 2011). Raw juice (∼151 L) was mechanically pumped into a 265 L juice tank where the juice was heated (∼1.3 ◦ C/min) to 80 ◦ C with 69 kPa steam and constant stirring. Temperature and pH of juice inside the tank were monitored using sensors. When the juice reached 80 ◦ C, it was immediately limed to the desired target pH with MOL under continuous mixing. Once the target pH was obtained the mixer was turned off and flocculant (StockhausenTM polyanionic solution 0.1%; 5 ppm) was added and stirred by hand using a paddle for 1 min. The flocculated, heated limed juice was then immediately gravity fed into a settling or clarification tank below and allowed to settle. The rate of settling was monitored via test port valves on the side of the clarification tank (Eggleston et al., 2011). A sample was collected in a test-tube at specified time intervals and settling visually observed in front of a bright lamp. The settling time was taken as the time for the mud to settle to the 25% volume capacity of the settling tank, i.e., at the #6 settling port valve. Mud was gravity drained from the bottom valve and stored in a −40 ◦ C freezer until analyzed. The CJ formed (∼114 L) was fed into pre-sterilized stainless steel holding buckets (19 L) that were closed with a lid, and stored overnight in a walk-in cooler at 4 ◦ C. The buckets and lids were pre-sterilized by treating with sodium hyperchlorite bleach

(3%) diluted 10-fold in water; the buckets and lids were then rinsed with water to remove any of the bleach solution and then allowed to air dry. The next morning the CJ was allowed to re-equilibrate to room temperature (∼25 ◦ C), then was subjected to UV light experiments when appropriate and stored (see Section 2.4). Duplicate aliquots (10 mL) were taken at 0, 2, 4, 20, and 144 h (6 days) for microbial and chemical analyses. The aliquots for chemical analyses were stored at −40 ◦ C until analyzed. 2.4. Pilot plant manufacture of heated (pasteurized) juice As the clarification process (a combination of heating and precipitation) was shown to stabilize and preserve sweet sorghum juice, the effect of heating alone was performed as a comparison to the combination of heating and precipitation. Heated juice was produced from sweet sorghum raw juice in the USDA-ARS-SRRC pilot plant (Eggleston et al., 2011) before the full clarification process occurred. Raw juice (∼151 L) was mechanically pumped into a 265 L juice tank where the juice was heated (∼1.3 ◦ C/min) to 80 ◦ C with 69 kPa steam and constant stirring. Temperature and pH of juice inside the tank were monitored using sensors. When the juice reached 80 ◦ C, the valve of the settling tank below was opened and ∼8 L was collected in pre-sterilized stainless steel buckets (19 L). The heated juice was kept in the buckets at room temperature for 30 min or the same amount of time that clarification settling took for the same juice following the method in Section 2.3. The heated juice was then similarly treated like the clarified juices, i.e., kept in pre-sterilized stainless steel holding buckets (19 L) with lids on and stored overnight in a walk-in cooler at 4 ◦ C and then the next day equilibrated to room temperature (∼25 ◦ C) stored at room temperature for 48 h. Duplicate aliquots (10 mL) were taken at 0, 2, 4, 24, and 48 h for microbial and chemical analyses. The aliquots for chemical analyses were stored at −40 ◦ C until analyzed. 2.5. Juice UV-C light experiments The raw or clarified juice was subjected to ultraviolet radiation (UV-C) in an in-line Aqua Ultraviolet SystemTM (Temecula, CA) at ambient temperature (∼25 ◦ C). This temperature was chosen as heating or cooling a UV system would be too expensive at the industrial site and because temperature has been reported to have little effect on UV-C treatment (Anon, 2013). At 25 ◦ C mesophilic microorganisms could grow if sufficient growth factors are present, depending on the microbial genus and species. The system consisted of a stainless steel concentric cylinder containing a horizontal UV lamp (15 W) centered inside, enclosed in quartz tube. The capacity of each unit was 2.1 L. Tubing connectors on the ends of the system allowed the juice to flow through clear, vinyl tubes (1.5 cm id) to create a circulation system. The juice was continuously re-circulated for different amounts of time. The juice flowing through the unit was kept constant at a flow rate of 681 L/h with an OASETM SP310G pump (OASE Living Water, Hermosa Beach, CA, USA). On arrival at USDA-ARS-SRRC in New Orleans, two raw juice aliquots (18 L each) placed in pre-sterilized stainless steel buckets (19 L), were filtered separately through window screen mesh to remove large fragments of plant matter or other large debris. One aliquot with the top covered was stored quiescently in the laboratory (∼25 ◦ C) while the second aliquot was pumped continuously through the UV-C system at 25 ◦ C. At 0, 2 and 4, and 20 h intervals after the initiation of UV-C light treatment, two aliquots (10 mL) were removed from the control and UV-treated raw sorghum juice samples and placed in 15 mL sterile conical centrifuge tubes. One tube of each sample type (control or UV-treated) was stored at −40 ◦ C for later chemical analysis while juice from the second tube of either sample type underwent microbial analyses. After 20 h, a

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final set of sample aliquots were collected with respective 10 mL aliquots stored at −40 ◦ C or analyzed for viable microbial populations. At this time, the UV light was turned off and both the remaining raw juice control and UV-treated raw juice were covered and stored quiescently in the lab (25 ◦ C) for up to 144 h (6 days) and analyzed again. CJs stored overnight at 4 ◦ C in pre-sterilized, closed (stainless steel) buckets (19 L) were allowed to equilibrate to room temperature (25 ◦ C) the next morning and then similarly treated with UV-C light like the raw juices, but were not initially filtered. After use, the UV light system was drained of sample and then pumped with a re-circulating anti-microbial solution (18.9 L, 10 ppm BusanTM , Buckman Laboratories, Memphis, TN) for approximately 1 h. After the anti-microbial solution was drained, clean tap water was constantly pumped through for 1 h with UV treatment to remove any trace of the anti-microbial compound and reduce the possibility of microbial contamination of the unit. After the water wash period, the tubing was removed from the unit to aid in the drying of the interior of the UV unit.

(Eggleston and Grisham, 2003), using CarboPac PA1 analytical and guard columns (Dionex Corp., Sunnyvale, CA, USA) at 25 ◦ C. Eluent conditions were: 100 mM NaOH (isocratic (0.0–1.1 min; inject 1.0 min), a gradient of 0–300 mM NaOAc in 100 mM NaOH (1.1–40.0 min), and return to 100 mM NaOH (40.1–45.0 min) to reequilibrate the column. Detector and auto-sampler conditions are stated in Eggleston and Grisham (2003). The samples were diluted 1 g/25 mL de-ionized water and filtered through a 0.45 ␮m filter. Glucose, fructose, and sucrose in juice samples were determined by IC-IPAD using the same columns following a 20 min, isocratic 100 mM NaOH method (Andrzejewski et al., 2013a,b).

2.6. Microbial analyses

3. Results and discussion

Raw, heated, and clarified sweet sorghum juices (control and UV-treated) were analyzed for viable bacterial populations immediately after sample collection. Serial dilutions with sterile water (1:10) were performed and aliquots (50 ␮L) spread on each of three Nutrient agar (Difco, Detroit, MI, USA) plates using a surface sterilized, bent glass rod to obtain total bacterial counts. Similarly, aliquots (50 ␮L) were spread on three MRS agar (EMD Millipore, Darmstadt, Germany) plates to obtain lactic acid producing bacterial (e.g., Lactobacillus sp., Pediococcus sp., Leuconostoc sp.) viable counts. The inoculated plates were incubated for 48 h at 30 ◦ C followed by enumeration of developed colonies. The next day, two 18.9 L aliquots were placed at room temperature with one serving as a control while the second was treated with UV light as described earlier. Duplicate aliquots (10 mL) were taken at 0, 2, 4, and 20 h from the control and UV-treated juices with one duplicate tube stored at −40 ◦ C while the other was analyzed for viable bacterial counts as described above. After the 20 h sample was collected, the UV was sanitized as described above. The remaining CJ juice control and UV-treated CJ were then stored quiescently with a cover for 6 days at room temperature. At the end of the 144 h (6 days) storage period, the sorghum juice (raw juice and CJ controls; UV treated raw juice and UV treated CJ) samples were analyzed for viable bacteria as described above.

3.1. Pilot plant clarification performance of sweet sorghum juices

2.7. Brix (percent dissolved solids), pH, color, and turbidity Brix was measured using an Index Instruments (Kissimmee, FL, USA) TCR 15–30 temperature controlled refractometer accurate to ± 0.01 Brix, and results expressed as an average of triplicates. Juice pH was measured on a Metrohm Brinkman 716 DMS Titrino (Riverview, FL, USA) with a Mettler Toledo (Columbus, OH, USA) xerolyte electrode. Nephalometer turbidity (NTU) measurements were taken on a Hach 2100 N turbidimeter (Loveland, CO, USA); results are an average of three measurements. Color of juices at pH 7.0 were measured as the absorbance at 420 nm and calculated according to the official ICUMSA method GS2/3-9 (1994). Samples (∼5 mL) were first diluted in triethanolamine/hydrochloric acid buffer (pH 7.0) and filtered through a 0.45 ␮m filter. 2.8. Sugars, sugar alcohols, and oligosaccharides measured using ion chromatography with integrated pulsed amperometric detection (IC-IPAD) An oligosaccharide fingerprint chromatogram (up to 12 dp) was obtained by using a strong NaOH/NaOAc gradient over 40 min

2.9. Statistics Turbidity, color, and microbial count data were statistically analyzed using PROC GLM in SAS 9.3 (SAS Institute, Cary, NC, USA). Means were separated using Duncan’s New Multiple Range Test.

The turbidity of raw and pilot plant clarified juices (CJs) was measured and results are listed in Table 1. Generally, the turbidity of the raw juices varied (P < 0.05) with cultivar, which is in agreement with previous results (Andrzejewski et al., 2013a,b). The turbidity of KN Morris raw juice was dramatically higher than for the other cultivars in the study, but this was most likely related to the freeze deterioration of this cultivar in the field which caused all the leaves to brown and senesce (Eggleston et al., 2010). Percent turbidity removal across clarification for all cultivars ranged from 92–98% which also agrees with Andrzejewski et al. (2013a,b) results. The turbidity of CJs varied significantly (P < 0.05) with cultivar, but values did not exceed 223 NTU. Although the relative high turbidity of the KN Morris raw juice increased settling time, turbidity removal was still 98.6% suggesting that freeze deteriorated juice can still be adequately clarified, but further studies are still needed to confirm this. Settling times were lower in the LA Theis and Top 76-6 juices, but it is not clear if this was because of differences between wholestalks and billets, an environmental effect, or slight variations in milling. Juice color was also measured because color may impede the penetration of UV-C light into the juice (Anon, 2013). As seen in Table 1, color values varied (P < 0.05) with cultivar and were higher (P < 0.05) in the juices from whole-stalks (LA) because of the higher amounts of green leaves. Although juice color often slightly decreased on clarification it occasionally slightly increased as well. Regardless, the color of clarified juices still remained at relatively high values. 4. UV-C radiation and clarification treatments on the stabilization and preservation of sweet sorghum juices Raw or clarified sweet sorghum juice was exposed to germicidal ultraviolet light (UV-C) from a 15 W lamp at 25 ◦ C, by passing it through a process tube at a constant flow rate. The effects of UV-C and clarification treatments on the total bacterial counts in raw juice from Theis (10 Sept; LA) stored at ambient temperature (∼25 ◦ C) are illustrated in Fig. 1. Before storage at 0 h, raw Theis juice contained total bacterial counts of 1.9 × 107 cfu/mL. This increased by 2-logs over the next 4 h of storage, subsequently stabilized, then slightly decreased by 144 h storage. The pH values of the raw juice similarly remained stable up to 20 h storage then decreased to pH 3.75 at 144 h (Fig. 2). The effect of UV-C light on raw juice did not ameliorate the bacterial counts

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Table 1 Pilot plant clarification of mature, sweet sorghum juices from Houma, LA and Delta BioRenewables, TN in 2013 (average of three replicates). Sweet Sorghum ProductionSite

Sweet Sorghum Cultivar

Harvest Method

Study Date

Time Settled (min)

Floc Size

Raw Juice Turbidity (NTU)

Clarified Juice Turbidity (NTU)

Turbidity Removal (%)

Raw Juice Color (ICU)

Clarified Juice Color (ICU)

Louisiana Louisiana Tennessee Tennessee Tennessee Tennessee

Theis Top 76-6 M81E Top 76-6 Top 76-6 KN Morris

Whole-stalk Whole-stalk Billet Billet Billet Billet

Sept 10 Sept 17 Sept 24 Oct 22 Nov 5 Nov 19

35 25 nd‡ 35 30 40†

small large nd‡ large large large

2600c 2200c 5400b 3333c 3300c 12,400a†

223b 175c nd‡ 115d 93e 172c

91.4 92.0 nd‡ 96.5 97.2 98.6

12292b 12726a 6794d 6144e 5491f 7647c

9564b 10839a nd‡ 5699e 6070d 5016f

‡ nd = not determined. Over limed due to pH electrode problems. *Different lowercase letters represent significant differences at the 5% probability level between samples in the same column for one date only. † Indications of field deterioration as pH lower on arrival. Only limed to pH ∼6.2

Fig. 1. Average total bacterial count in sweet sorghum juice from Theis cultivar harvested and processed on Sept 10, 2013 in Louisiana.

but caused them to be even slightly worse (Fig. 1). In dramatic contrast, clarification achieved a 3-log reduction in the initial (0 h) total bacterial counts in the Theis raw juice. Like for raw juice, CJ also subjected to UV-C light stored even worse than the CJ control (Figs. 1 and 2). The recycling of juice by pumps will have allowed for better mixing of the microbes and for the maximum utilization of microbial growth nutrients, i.e., it almost acted as a fermenter itself; hence, the slightly worse data with UV treatment over the controls. Total bacterial counts for Top 76-6 (17 Sept; LA) stored juices are illustrated in Fig. 3A and lactic acid bacteria counts in Fig. 3B. The 0 h control and UV-treated raw juices contained 1.6 × 105 and 4.2 × 107 cfu/mL total bacteria, respectively (Fig. 3A). Such high populations of bacteria would adversely impact the formation of end-products via fermentation by competing with the added fermentation organism of choice for valuable nutrients. Different cultivars of sweet sorghum contain different sugar compositions and will be expected to maintain different amounts and types of flora microorganisms. Both control and UV-treated raw juices followed very similar total bacterial growth patterns with time (Fig. 3A). Like Theis on Sept 10 (Fig. 1), the clarification process achieved a marked reduction in the total bacterial counts in the 0 h CJ to a negligible 10 cfu/mL. Moreno et al. (2012) reported that clarification of sugarcane juice removed microbes (bacteria and yeast), and was even better than microfiltration. The growth of bacteria across 20 h storage increased more considerably and faster in the UV-treated than control CJ, although by 144 h, 109 total bacteria occurred in both CJs (Fig. 3A). Clarification also reduced the amount of lactic acid bacteria to ∼10 cfu/mL in the Top 76-6 (Sept 17) juice (Fig. 3B). However, unlike the total bacteria, lactic acid bacteria did not increase in the CJ control for up to 20 h of storage

at 25 ◦ C, indicating the clarification process reduced lactic acid bacterial populations better than for total bacteria. Similar to the total bacterial counts (Fig. 3A), however, the UV-treated CJ followed the same growth path as the control and UV-treated raw juice. At 0 h, M81E and Top 76-6 raw juices from TN (24 Sept and 22 Oct), contained 2.8 × 108 and 3.1 × 107 cfu/mL, respectively (Table 2). Concomitantly, there were 4.5 × 107 and 2.9 × 106 cfu/mL of lactic acid bacteria in 0 h M81E and Top 76-6 raw juices, respectively (Table 2). Deaeschel et al. (1981) earlier reported that fresh sweet sorghum juice contained 108 microorganisms per mL which were mainly hetero-fermentative Leuconostoc mesenteroides lactic acid bacteria and Gram-negative rods, with some Lactobacilli, yeasts, and non-fecal coliform bacteria. In this study, the effect of clarification at 0 h was to dramatically reduce the total bacterial counts by 3- to 4-logs, although the decrease varied with sample date and cultivar. This reduction was even greater, i.e., 6-logs, for lactic acid bacteria counts which were not even detected in the Top 76-6 (22 Oct) clarified juice (Table 2). In general, for untreated raw juices the total bacterial counts increased by 1- to 3-logs during the 20 h storage period at 25 ◦ C, with most growth occurring between 4 and 20 h. Between 20 and 144 h storage, the total bacterial counts consistently decreased approximately 4- to 20-fold and the Brix and pH concomitantly decreased (Table 2 and Fig. 2). Furthermore, after 144 h of storage the raw juices often (i) appeared viscous because of extracellular polysaccharides, i.e., dextran from Leuconostoc mesenteroides, (ii) had bubbles, and (iii) smelled of alcohol. Except for Theis on 10 Sept, the lactic acid bacteria also decreased markedly in the raw juices from 20 to 144 h (Fig. 3 and Table 2). These results suggest that other types of microorganisms took over the microbial deterioration of the juice by day 6 (144 h) of storage. Wu et al. (2010) observed that after 1 week of storage at 25 ◦ C, more than 95% of bacteria in the sweet sorghum juice was homo-fermentative compared to the hetero-fermentative lactic acid bacteria in the first week of storage. However, in this study the lower Brix values and visual characteristics of the 144 h stored juices indicated possible yeast deterioration with the formation of large amounts of carbon dioxide and ethanol. Overall, the UV-C treatment (15 W; 25 ◦ C) did not act as a bactericide and frequently made juice deterioration slightly worse. The relatively high Brix, turbidity, and color values of the sweet sorghum juices (Table 1) most likely impeded the surface action and penetration of the UV-C light in the whole volume of juice (Anon, 2013). Maple saps, in contrast, are low Brix and often colorless. The 15 Watt UV-C aquaculture system we used may not have had sufficient energy and duration needed to inactivate the microorganisms and disinfect the whole liquid (Knight, 2013). In strong contrast to the UV-C treatment, clarification of the juice dramatically improved the stability and storage of the four sweet sorghum juices from all cultivars studied.

G. Eggleston et al. / Industrial Crops and Products 64 (2015) 258–270 Raw Juice 8

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Fig. 2. The change in pH over storage time of UV-C and clarified treated raw sweet sorghum juices compared to no treatment.

Fig. 3. Average (A) total bacterial count, and (B) lactic acid bacterial count in sweet sorghum juice from Top 76-6 cultivar harvested and processed on Sept 17, 2013 in Louisiana.

5. Ion chromatography fingerprint analyses Wu et al. (2010) reported that at room temperature (∼25 ◦ C) the sugar content and profile of sweet sorghum juice changed dramatically during 15 days storage. Sucrose content decreased rapidly during storage and essentially disappeared after 5 days whereas glucose and fructose did not change markedly (Wu et al.,

2010). Ethanol, lactic, formic and acetic acids appeared after 5 days at room temperature (25 ◦ C). Daeschel et al. (1981), Wu et al. (2010), and Lingle et al. (2013) also reported that sweet sorghum juice was susceptible to hetero-fermentative lactic acid bacteria, in particular Leuconostoc mesenteroides, which is also the major cause of microbial deterioration in sugarcane (Eggleston, 2002) and sugar beet juices (Eggleston and Huet, 2012). Eggleston and

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Table 2 Total and lactic acid bacterial counts in raw and clarified M81E (24 Sept, 2013) and Top 76-6 (22 Oct, 2013) sweet sorghum juices, treated with and without UV-C light, on storage at 25 ◦ C (average of three replicates). Juices were obtained from Delta BioRenewables, Memphis, TN. Storage time (h)

Raw Juice (control)

UV-treated raw juice

Clarified juice (control)

UV-treated clarified juice

Total Bacteria Counts ± Std. Dev (cfu/mL) (Nutrient Agar) M81E; 24 Sept, 2013 0 2 4 20 144 0 2 4 20 144

2.8 × 108 6.7 × 107 1.1 × 108 1.1 × 108 1.5 × 108 6.2 × 107 3.2 × 108 3.1 × 109 1.2 × 107 1.2 × 106 Top 76-6; 22 Oct, 2013 3.1 × 107 2.9 × 107 2.9 × 107 2.9 × 107 2.8 × 107 2.9 × 107 2.3 × 108 3.8 × 108 4.3 × 107 4.2 × 107

Raw Juice (control)

UV-treated raw juice

Clarified juice (control)

UV-treated clarified juice

Lactic Acid Bacteria Counts ± Std. Dev (cfu/mL) (MRS Agar)

na* na na na na

na na na na na

4.5 × 107 3.3 × 107 4.8 × 107 2.5 × 108 9.8 × 106

9.7 × 107 3.7 × 107 9.3 × 107 5.6 × 109 2.9 × 106

na na na na na

na na na na na

2.1 × 103 2.5 × 103 4.2 × 104 3.4 × 103 3.6 × 108

6.2 × 103 2.1 × 105 1.7 × 105 2.4 × 109 3.0 × 108

2.9 × 106 3.5 × 106 3.6 × 106 2.5 × 108 3.9 × 107

3.1 × 106 3.1 × 106 3.1 × 106 4.0 × 108 4.5 × 107

0 0 0 5.5 × 103 2.8 × 108

0 9.3 × 104 1.7 × 105 3.0 × 109 ×108

* na = not applicable.

Fig. 4. IC-IPAD fingerprint oligosaccharide chromatograms of stored Theis sweet sorghum juice from Louisiana on Sept 10, 2013. (A) Raw juice and (B) UV-treated raw juice. Time of storage and Brix of the juices are denoted on the overlaid chromatograms. G = glucose, F = fructose, and S = sucrose.

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Fig. 5. IC-IPAD fingerprint oligosaccharide chromatograms of stored Top 76-6 sweet sorghum juice from Tennessee on Oct 22, 2013. (A) Raw juice and (B) clarified juice, and (C) clarified juice treated with UV-C light. Time of storage and Brix of the juices are denoted on the overlaid chromatograms.

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Grisham (2003) previously reported the use of an ion chromatography with integrated pulsed amperometric detection (IC-IPAD) method using a strong acetate gradient to create oligosaccharide fingerprint chromatograms, to successfully demonstrate the deterioration of sugarcane juice. This IC-IPAD method was applied to Theis, Top 76-6, and M81E cultivars from both LA and TN juices that had been treated with UV-C irradiation or clarification, and typical chromatograms are shown in Figs. 4 and 5. For all four sampling dates studied, deterioration products were only visible on the IC fingerprint profiles after 20 h storage, although there was variation in the amount and type of deterioration products that formed. Leuconostoc deterioration products mannitol, leucrose, isomaltose, and isomaltotriose (Eggleston, 2002; Eggleston and Huet, 2012) were formed early in the deterioration process, strongly indicating that hetero-fermentative lactic acid bacteria were mostly (but not solely) responsible for early deterioration. Similar to the raw juice, IC-IPAD deterioration products were not visible in UV-treated raw juice until 20 h storage. Although deterioration products were visible at 20 h, the Brix hardly changed (Figs. 4 and 5) which indicated that juice solids were only transformed into solid deterioration products. After 144 h storage, numerous deterioration products were detected in all treated and untreated samples, including a series of malto oligosaccharides and ethanol. The Brix was always markedly reduced (Figs. 4 and 5), indicating other microorganisms that form gases or other compounds which vaporize were causing juice spoilage, i.e., yeast that form high amounts of ethanol and carbon dioxide. Furthermore, considerable losses in total fermentable sugars (glucose + fructose + sucrose) occurred in all the samples, ranging from 28 to 97% depending on cultivar and treatment. UV-C treated juices incurred more total sugar losses than their respective controls. Also, in general, sucrose disappeared more rapidly than glucose and then fructose. Clarification dramatically reduced the formation of deterioration products in Top 76-6 (Oct 22) juices, which is illustrated in Fig. 5. Unlike for the raw juice (Fig. 5A), deterioration products were not visible in the CJ even after 20 h storage, which agrees with the microbiology data (Table 2). However, like raw juice stored for 144 h (6 days), the CJ stored for 144 h contained numerous deterioration products and had considerable losses in fermentable sugars (results not shown), indicating that the CJ had spoiled between 20 and 144 h; this again confirmed the microbiology data (Fig. 2). The effect of UV-C irradiation on the CJ was negligible with respect to improving the stability of the juice and actually made it worse on some occasions (Fig. 5 C). This is further evidence that the UV-C light system (15 W) in this study at 25 ◦ C did not act as an anti-microbial device because the relatively high color, Brix, and turbidity of all the sweet sorghum juices did not allow the light to affect the surface or penetrate the interior of the juice.

6. Heat pasteurization treatment As clarification was shown to dramatically improve the preservation of sweet sorghum juice, further experiments were conducted to elucidate if it was the heating component of the clarification processes that imparted preservation, the precipitation component, or both components. The first part of the clarification process involves the heating of the juice to 80 ◦ C. Next MOL and then flocculant is added and the juice is allowed to settle in a tank. During settling, impurities are precipitated. The time for settling was approximately 30 min settling period, but depends on the cultivar (Table 1; Andrzejewski et al., 2013a,b). Thus, the heating component of the clarification process is essentially pasteurization at 80 ◦ C for 30 min. Kumar et al. (2013) studied the laboratory pasteurization of sweet sorghum juice 70, 80, and 90 ◦ C for 10, 5, and 2 min,

Fig. 6. Average total bacterial and lactic acid bacteria counts in raw, heated, and clarified sweet sorghum juices from (A) Top 76-6 cultivar harvested and processed on 5 Nov, 2013, and (B) KN Morris cultivar harvested and processed on 19 Nov, 2013. Both juices were extracted at Delta BioRenewables in Memphis, TN.

respectively, that were then stored at 35, 40, and 45 ◦ C for up to 21 days. Although the heat broke down sucrose into glucose and fructose, the glucose and fructose did not decrease and, therefore, fermentable sugar content was maintained. Superior preservation occurred in the 90 ◦ C pasteurized juices; however, the rate of heating was not stated (Kumar et al. (2013) and microbial growth not reported. Pasteurization is a widely practiced preservation method employed by the food industry since heating liquid foods at high temperature kills a major fraction of microorganisms. In this study, we compared the storage of raw, heated, and clarified juice from Top 76-6 and KN Morris cultivars (TN) over 48 h at 25 ◦ C. These samples were not subjected to UV-C treatment due to the previous unsuccessful results. The effect of heating alone was to reduce turbidity by 25.2 and 84.9% in Top 76-6 and KN Morris juices, respectively. This, however, was still not as much as clarification which reduced Top 76-6 juice turbidity by 97.2% (final CJ turbidity was 93 NTU) and KN Morris by 98.6% (CJ was 172 NTU). These results are in agreement with those of Andrzejewski et al. (2013a,b). Even though there was 10.6% greater color in Top 76-6 heated than raw juice, the color of the CJ was the same as the heated juice (6072 ICU). In comparison, KN Morris color decreased 30.7% on heating, i.e., from 7647 to 5299 ICU, which decreased by a further 4.1% to 5084 ICU in the CJ. Before storage, both Top 76-6 and KN Morris raw juices (0 h) contained 4.1 × 107 and 2.8 × 108 total bacteria per mL, respectively (Fig. 6). The total bacteria count in the KN Morris juice was the highest detected in this study, and may be because of the considerably higher turbidity levels caused by an excess of brown leaves (Table 1). This is further evidenced by the existence of a

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Fig. 7. IC-IPAD fingerprint oligosaccharide chromatograms of stored Top 76-6 sweet sorghum juice from TN on 5 Nov, 2013. (A) Raw juice, (B) heated juice, and (C) clarified juice. Time of storage and Brix of the juices are denoted on the overlaid chromatograms.

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Fig. 8. IC-IPAD fingerprint oligosaccharide chromatograms of stored KN Morris sweet sorghum juice from TN on 19 Nov, 2013. (A) Raw juice, (B) heated juice, and (C) clarified juice. Time of storage and Brix of the juices are denoted on the overlaid chromatograms.

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very strong correlation (R2 = 0.967; y = 27309x-5E + 07) between the total bacteria count (y axis) and raw juice turbidity (x axis) for all the samples in this study. Eggleston et al. (2014a,b) reported that microbial bodies contribute to the turbidity of sugarcane juices. Total bacterial counts in raw juices from both cultivars during the 48 h storage period at 25 ◦ C increased ∼5-fold, with most growth occurring between 4 and 24 h (Fig. 6). Total bacteria in the 0 h heated juices from both cultivars were approximately 4-log less than the 0 h raw juices, but after 48 h storage contained slightly more, with KN Morris heated juice worse than Top 76-6 heated juice (Fig. 5). Similar to the previous clarification experiments (Figs. 1 and 3; Table 2), CJ from both cultivars at 0 h contained 4-log less total bacteria and no detectable lactic acid bacteria (Fig. 6). This confirms that the clarification process destroyed or removed all lactic acid bacteria, although other bacteria remained at ∼103 cfu/mL levels. As the 0 h total bacterial counts in the heated juices were very similar to those in the CJs, this indicated that the heat component of clarification was mostly responsible for the reduction in total bacteria. However, although no lactic acid bacteria were detected in both the 0 h heated and clarified juices of Top 76-6, a small amount of lactic acid was detected in the heated juice of KN Morris, but none in its CJ. This suggests that heating may not be quite as efficient as clarification in removing lactic acid bacteria. There was no significant growth in the amount of total bacteria in the CJ across the 48 h storage period. Only small amounts of lactic acid bacteria were detected at 48 h in the KN Morris CJ, which were produced between 24 and 48 h because none were detected at 24 h (Fig. 6). Up to 24 h storage, similar to the clarification process there were no lactic acid detected in Top 76-6 heat pasteurized (80 ◦ C; 30 min) juices. Also similar to the CJs, lactic acid bacteria were only detected in low levels in the 48 h samples (Fig. 6A). Lactic acid formation was slightly worse in KN Morris heated than CJ, but even at 48 h only a negligible 4.7 × 102 /mL of lactic acid bacteria were detected in the heated juice which was still considerably lower than the raw juice. Thus, both heating and clarification stabilized and preserved the juice compared to raw juice, with clarification slightly better than heating alone. The removal of suspended and turbid solids during the clarification precipitation process may have (i) reduced the availability of some macro and micronutrients for bacterial growth, (ii) reduced surface area of particles for microbes to grow on, or (iii) precipitated out damaged/vegetative microbial bodies and spores in the mud. The effects of heat pasteurization (80 ◦ C; 30 min) on oligosaccharide fingerprint analyses for both Top 76-6 (Nov 5) and KN Morris (Nov 19) juices are illustrated in Figs. 7 and 8, respectively. No formation of deterioration products was detected in Top 76-6 heated and CJs, stored from 0 to 48 h at 25 ◦ C (Fig. 7). Similar results were found for the KN Morris heated and CJs, although a slight amount of oligosaccharides after the sucrose peak were detected in the 24 and 48 h samples (Fig. 8). This agrees with the lactic acid microbial data in Fig. 6B, which showed that ∼103 cfu/mL lactic acid occurred in the 24 and 48 h KN Morris heated juice samples. Furthermore, after 28 h of storage, for KN Morris heated juice, there was a 7.2% decrease in total fermentable sugars with a 16.3% loss of sucrose into glucose and fructose, while for CJ there was no loss of total fermentable sugars. This was in dramatic comparison to raw juice after 48 h, which lost 29.2 and 69.7% of total fermentable sugars and sucrose, respectively. No marked changes in Brix (Figs. 7-8), and pH (results not shown) were noted across the 48 h storage periods for both heated and clarified juices. This is in strong contrast to the raw juices where there was a slight decrease in Brix (Figs. 7-8), and pH decreased from 4.94 to 3.67 and 5.01 to 3.83 in the 0 and 48 h Top 76-6 and KN Morris raw juices, respectively. In comparison, deterioration products were found in the 24 h stored raw juice samples from both cultivars, which were

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worse after 48 h storage (Figs. 7-8). Thus, the IC fingerprint profiles confirmed the bacterial growth results shown in Fig. 5. Overall, these results also show that the IC-IPAD fingerprint method can detect >102 cfu/mL of lactic acid bacteria growth. The effect of heat pasteurization was very similar to the whole clarification process which confirmed that heat was the major contributor to the juice stabilization. 7. Conclusions The storage of raw juice extracted from sweet sorghum poses a great technical challenge. Raw juices contained very high populations of bacteria up to 109 total bacteria cfu/mL. Such high populations of contaminating bacteria would detrimentally impact the formation and yields of end-products via fermentation, by competing with the added fermentation organism of choice for valuable nutrients. Storage at room temperature (∼25 ◦ C) caused sweet sorghum juice to spoil between 4 and 20 h. UV-C treatment (15 W; 25 ◦ C), used to preserve low Brix and colorless maple saps, did not improve spoilage. This was most likely because of insufficient energy as well as the relative high Brix, turbidity, and color of sweet sorghum juices impeding the penetration of the light into the juice. In strong contrast, clarification of sweet sorghum juice dramatically improved the storage of juice at 25 ◦ C, and significantly (P < 0.05) reduced both the total and lactic acid bacteria counts, with the lactic acid bacteria being reduced to negligible levels. The heating component (80 ◦ C; 30 min) of the clarification process was mostly responsible for reducing juice spoilage, but stability and preservation were slightly better with the subsequent precipitation component. Moreover, clarified juice and juice heated at 1.3 ◦ C/min to only 80 ◦ C and kept for 30 min, allowed sweet sorghum juice to be stored for at least a 48 h. In contrast, milk is high-temperature, short time (HTST) pasteurized by heating to 72 ◦ C for 15 sec in a plate heater, which reduces total bacteria by 2-logs (Anon, 2009). A higher heating rate would ward off loss of fermentable sugars in sweet sorghum juices to acid degradation reactions. HTST milk has a refrigerated shelf-life of 2–3 weeks. Further studies will now be undertaken to investigate the maximum storage period and the effect of the rate of heating on sweet sorghum juice stability. Sweet sorghum juice can also be extracted with diffusion technology at juice temperatures below 72.4 ◦ C (gelatinization temperature of sweet sorghum starch; Alves et al., 2014). The diffusion heat may help to stabilize the juice for a few hours, but research needs to verify this. Research will also be conducted on how the degree of juice deterioration determines the degree of fermentation yields of different end products. This needs to be evaluated for both yeast based and bacterial biocatalysts. Acknowledgements The authors thank Larry Boihem for help in obtaining sweet sorghum juice from Tennessee, and are grateful to the United Sorghum Checkoff Program for funding this research. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. References Alves, F., Polesi, L., Aguiar, C., Sarmento, S., 2014. Structural and physicochemical characteristics of starch from sugarcane and sweet sorghum stalks. Carbohydr. Polym. 111, 592–597. Andrzejewski, B., Eggleston, G., Lingle, S., Powell, R., 2013a. Development of a sweet sorghum juice clarification method in the manufacture of industrial feedstocks for value-added products. Indust. Crops and Products 44, 77–87.

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