Research Article Cite This: ACS Sustainable Chem. Eng. 2019, 7, 12240−12247
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Using Zeolites To Protein Stabilize White Wines Agnieszka Mierczynska-Vasilev,*,†,‡ Satriyo K. Wahono,‡,⊥,§ Paul A. Smith,†,∥ Keren Bindon,† and Krasimir Vasilev⊥
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The Australian Wine Research Institute, Waite Precinct, Hartley Grove cnr Paratoo Road, PO Box 197, Urrbrae, Glen Osmond, Adelaide, South Australia 5064, Australia ‡ Future Industries Institute, University of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, South Australia 5095, Australia ∥ Wine Australia, Industry House, National Wine Centre, Cnr Botanic & Hackney Roads, PO Box 2733, Kent Town, Adelaide, South Australia 5000, Australia ⊥ School of Engineering, University of South Australia, Mawson Lakes Campus, Mawson Lakes, Adelaide, South Australia 5095, Australia § Research Unit for Natural Product Technology, Indonesian Institute of Sciences, Jl. Jogja-Wonosari Km 31.5 Gading, Playen, Gunungkidul, Yogyakarta 55861, Indonesia, PO BOX: 174 WNO S Supporting Information *
ABSTRACT: The use of bentonite fining in the global wine industry causes losses in the range of US$1 billion per year. This work explores for the first time the potential use of natural zeolites as an alternative to bentonite to remove haze proteins from white wines. Wines treated with zeolites were compared to those obtained after fining using two commercial bentonites (Vitiben and Pluxcompact). Semillon wine was fully stabilized by applying 4 g/L of zeolite, whereas Sauvignon Blanc and Chardonnay required 6 g/L dosage of zeolite. Analysis of metal content showed that zeolites reduced potassium concentration by more than 30% in the treated wines, which could lead to improved tartrate stability. Importantly, compared to bentonites, zeolites cause much less wine loss, and they can be potentially reused as soil amendments in agriculture. The results of this study indicate that natural zeolites can offer winemakers an alternative to bentonite for haze protein removal from white wines. KEYWORDS: Natural zeolites, Haze, Haze-forming proteins, Protein removal, White wine, Potassium ion decrease
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Bentonite9 is a montmorillonite clay mineral having hydration and swelling properties. Once the bentonite has been prepared in the form of a slurry, it is mixed with the wine and left to settle. In the final step the wine is separated from the bentonite lees by racking or filtration.8 Bentonite fining does have some disadvantages. One of bentonite’s drawbacks is that it removes some of the flavor and aroma compounds in wine.3,10,11 Further disadvantages of bentonite fining are wine volume loss (3−10%) because of poor settling and swelling2 and high disposal costs.10,12 This, coupled with transportation, handling, and disposal, leads to an annual estimated loss of close to US$1 billion worldwide.13 To date, several alternative techniques to bentonite fining have been studied. Those include ultrafiltration,14 flash pasteurization,15 proteases,16,17 chitosan,18 carrageenan and pectin,19−21
INTRODUCTION Improper storage conditions of finished white wine can lead to the formation of wine turbidity or sediments.1 On the one hand, wine proteins can precipitate and cause turbidity if bottled wine is exposed to high temperatures during wine storage or transport.2 On the other hand, potassium bitartrate crystals may form when bottled wine is exposed to low temperatures.3 Wine turbidity or precipitation in bottled wines can lead to negative sensory consequences, but more often it simply affects consumer responses.4,5 Unlike hazy wine, stabilized wines show no turbidity or sediments during wine handling or storage. As a consequence, winemakers stabilize their wines, when necessary, to avoid the formation of hazes and deposits. However, excessive stabilization can reduce the quality of the wine. All grapes contain proteins sensitive and insensitive to heat.6 The amount of heat-sensitive protein varies according to the vintage, grape variety, grape maturity, and pH.7 The stabilization of wine proteins involves determining the degree of heat sensitivity and elimination of heat-sensitive proteins8 using bentonite. © 2019 American Chemical Society
Received: March 19, 2019 Revised: June 3, 2019 Published: June 11, 2019 12240
DOI: 10.1021/acssuschemeng.9b01583 ACS Sustainable Chem. Eng. 2019, 7, 12240−12247
Research Article
ACS Sustainable Chemistry & Engineering zirconia,22 and haze protein removal via plasma coated magnetic nanoparticles.23,24 Zeolites25,26 are crystalline, hydrated aluminosilicates of sodium and calcium, and to a lesser extent barium, strontium, potassium, magnesium, and manganese. Zeolites are characterized by the presence of so-called zeolitic water in their composition, which is removed from its structure during heating and leaves the open skeletal structure of the crystal unchanged. A structure like this offers unique molecular sieve, sorption, and ion exchange properties that have been applied in many aspects of life: from water and sewage treatment (removal of ammonium ions, radioactive elements,27 heavy metals,28 oil pollution) to adsorption of water29,30 and gases,31,32 as well as in agriculture and other types of industry. In 2010, Mercurio et al. used zeolitized tuffs to treat and stabilized Italian wines from the Campania region.33 As a result of their widespread application, natural zeolites are referred to as “magic rock” by researchers and mineralogists.34 Research is in progress to enhance the use of zeolites.35−37 Because of the excess negative charge on the surface of zeolites38 they are an excellent candidate for the removal of proteins from wines. In contrast to bentonite, zeolites are free of shrink−swell behavior. The objective of this study was to examine the capacity of natural zeolites to protein stabilize white wines and to test the impact of zeolite treatment on wine quality. Ultimately, the goal is to develop an economically and technically feasible alternative to bentonite for removing haze proteins from wine.
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reported as the difference between the two, in nephelometric turbidity units (NTU). Wines with ΔNTU < 2.0 between heated and unheated samples were considered heat-stable. Organic Acids. Concentrations of organic acids in wines before and after treatment with natural zeolites and bentonites were measured by HPLC Agilent Technologies (1200 Series) as described previously by Marangon et.al.22 Phenolic Content. The phenolic content of wines before and after treatment with natural zeolite and bentonite was measured using a Cary 60 UV−vis spectrometer (Agilent Technologies). Metals Analysis. Metals content in wine was determined by inductively coupled plasma−optical emission spectrometry (ICPOES) performed by AWRI Commercial Services. Settling Experiments. Natural zeolite and bentonite suspensions were prepared by mixing the mineral slurry with white wine for 3 h. The suspensions were then transferred to 100 cm3 graduated glass cylinders and allowed to settle for 48 h. After this time the volume of lees created in the graduated measuring cylinders was measured using a caliper. Protein Modeling. Structures of Chitinase (CHI) and thaumatinlike protein (TLP) was modeled using the Swiss-Model. The structure and the surface of the protein were presented using UCSF Chimera 1.13.
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RESULTS AND DISCUSSION Characterization of Natural Zeolite. Surface properties and structure of the natural zeolite used in this work are listed in Table 1. The bulk and surface elemental composition are Table 1. Characterization of Natural Zeolite Sample specific surface area pore volume average pore diameter micropore surface area external surface area (ESA) structure by XRD
MATERIALS AND METHODS
Natural Zeolites. Samples used for the present research came from Indonesian mining company (CV Mountain Stones) in a powder form. The particle size distribution was determined by a Sieve Shaker (AS 200). Inductively coupled plasma−mass spectrometry (ICP-MS) and energy dispersive X-ray spectroscopy (EDX) were used to determine the bulk elemental composition of zeolites. Their surface composition was analyzed by X-ray photoelectron spectroscopy (XPS). The low-temperature nitrogen adsorption/desorption isotherms (77.15 K) were recorded using an ASAP 2420 adsorption analyzer (Figure S1). The specific surface area and porosity were obtained from the standard BET (Brunauer−Emmett−Teller) model and the BJH (Barrett−Joyner−Halenda) model, respectively. A total of 250 mg of zeolite samples were used for the BET analysis. The morphology of natural zeolites was examined by means of scanning electron microscopy (Hitachi SU3500). In this work zeolites were used as received (in text referred to as dry zeolites) and in the form of a slurry (in text referred to as wet zeolites). Bentonites. Two commercially available bentonites were used for comparison purposes: Vitiben and Pluxcompact. Bentonite stock solutions were prepared according to the suppliers’ instructions and rehydrated for 3 h. White Wines. Three unfined white wines were used, which were produced according to the usual methods of commercial wine production. Sauvignon Blanc (SAB), Semillon (SEM), and Chardonnay (CHA) were supplied by Accolade Wines, Reynella, South Australia. Before experiments were undertaken, the wines were stored below 10 °C. Table S1 (Supporting Information) presents basic chemical data characterizing the three used wines. Protein Content. The concentration of wine proteins was measured by HPLC (Agilent Technology) according to the previously published method.23 Heat Test. Wines were heated at 80 °C for 2 h, then cooled on ice for 2 h, and analyzed after equilibration to room temperature. The turbidity of the wine before heating and after heating and cooling was measured for each wine using a nephelometer, and the result was
a
25.95 m2/g 5.31 × 10−2 cm3/g 8 nm 8.18 m2/g 17.77 m2/g calcite Ca(CO3) and mordenite (Na8(Al8Si40O96)· 24H2O)a−clinoptilolite ((Na4K4)(Al8Si40O96)·24 H2O)a
Representative unit cell formula taken from ref 25.
shown in Table S2 together with the Si/Al ratio. The ratio of silica to alumina in the crystal structure of zeolite is an important characteristic of this mineral.39,40 Generally, as the Si/Al ratio increases, the thermal stability, acid strength, and hydrophobicity increase and ion-exchange capacity decreases.39 Depending on the Si/Al composition, zeolites are classified as follows: low Si/Al zeolites (Si/Al ratio of less than 2), intermediate Si/Al zeolites (Si/Al = 2−5), and high Si/Al zeolites (Si/Al > 5). In the current work, an intermediate Si/Al zeolite (Si/Al ≅ 4) was used. The XRD analysis showed that the sample had calcite and mordenite−clinoptilolite structure, as presented in Figure S3. The SEM micrographs are shown in Figure S4. In addition, the zeolite was ground to five different size ranges (shown in Figure S2) to determine the effect of particle size on protein removal efficacy. Adsorption Mechanism of Haze-Forming Proteins on Natural Zeolite. The aim of this research was to investigate adsorption capacity of natural zeolites to protein stabilize white wines. The initial protein concentration in three investigated wines was analyzed by high-performance liquid chromatography (HPLC). Figure S5 contains the HPLC chromatograms of control wine samples and after treatment with 10 g/L of natural zeolite. The initial protein concentrations in Sauvignon 12241
DOI: 10.1021/acssuschemeng.9b01583 ACS Sustainable Chem. Eng. 2019, 7, 12240−12247
Research Article
ACS Sustainable Chemistry & Engineering
adsorbed onto zeolites by cation exchange between the basic amino acid side chains of the proteins (represented in red), such as lysine and arginine, and ions such as K+, Na+, or Ca2+ located on the zeolite surface or/and in the zeolite channels. Influence of Zeolite Particle Size on Protein Removal Capacity. The effect of zeolite particle size on protein removal capacity was investigated. Given that during wine fining with bentonite a slurry is added to unfined wine for efficient protein recovery, the impact of zeolite rehydration on protein removal efficiency was also investigated. Zeolites were used in a dry state (Figure 3A) and after rehydrating in Milli-Q water for 3 h at 1:10 zeolite:water ratio (Figure 3A). The initial protein concentrations of the unfined Sauvignon Blanc, Semillon, and Chardonnay wines were 182, 165, and 103 mg/L, respectively. Wet zeolites were found to remove more protein from wine than dry zeolites for all particle size fractions. In terms of particle size, the 50−20 μm fraction was the most effective for both wet and dry zeolite treatments. In general, particle size had a significant influence on protein removal from wines. The protein concentration was 4, 3.3, and 2.4 times lower after treatment using the fine zeolite fraction (50−20 μm) compared to coarse zeolite fraction (420−300 μm) in the SAB, SEM, and CHA wines, respectively. This was expected as the surface area of smaller particles is significantly greater than those of bigger particles. It was also observed that rehydration of the 50−20 μm size fraction zeolite improved protein removal by 55, 60, and 35% for the SEM, SAB, and CHA wines, respectively. Hydration of the zeolites proved to be beneficial for protein removal, so the influence of the time of hydration and the treatment duration was further investigated. The hydration times compared were 1 and 3 h, and the treatment durations trialled were 0.5, 1, 3, and 6 h. Zeolites with particle sizes of 50−20 μm were selected for these experiments as they had been shown to be the most efficient in removing haze proteins (Figure 3). Studies were carried out with Sauvignon Blanc as this wine had the highest initial protein concentration. As shown in Figure 4A, the original total protein concentration sharply decreased from 182 mg/L to below 80 mg/L within 1 h and continued to decrease gradually up to 3 h of treatment. Longer treatment time did not result in further reduction in protein concentration. Three hours of hydration led to slightly better results than 1 h, reducing the protein concentration to about 50 mg/L after 3 h of treatment. The change in concentration of TLPs and CHI individually is shown in Figure 4B,C, respectively. The concentrations of both proteins decreased in a similar way to that observed for the total protein concentration (Figure 4A). However, while TLPs reached a plateau after 3 h, the concentration of CHI slightly increased after 3 h of treatment. This suggests that for CHI desorption processes may occur after 3 h of adsorption, and a longer treatment time is not advisible since the process may be less effective. From another perspective, this finding may offer the opportunity to fine-tune the concentration of TLPs and CHI in wines, which may lead to interesting sensory consequences. Effect of Zeolite Concentration on Wine Protein Removal Capacity and Heat Stability. Three unfined white wines (SAB, SEM, and CHA) were treated with hydrated natural zeolites (50−20 μm size fraction) at four different dosage rates of 2, 4, 6, and 8 g/L. The wines were treated with the zeolites for 3 h. Figure 5a shows that 2 g/L of zeolite was sufficient to reduce the protein concentration by 50%, 40%,
Blanc (SAB), Semillon (SEM), and Chardonnay (CHA) wines were 182, 165, and 103 mg/L, respectively. Whereas in Sauvignon Blanc and Semillon wines both chitinases and thaumatin-like proteins (TLPs) were present, there was no chitinases in Chardonnay wine. After treatment with 10 g/L of natural zeolite the pathogenesis-related proteins were selectively captured and completely removed. Figure 1 shows a model which represents a threedimensional (3D) structure of Chitinase M1 protein and
Figure 1. Hydrophobic surface of (A) CHI M1 protein and (B) TLP N1 protein modeled with Chimera. The backbone is depicted in yellow, the positively charged amino acids (Arg, His, and Lys) are in red, and the negatively charged amino acids (Asp and Glu) are in blue.
thaumatin-like N1 protein. The distribution of basic and acidic amino acid residues is highlighted. These 3D models were produced with Chimera software using data from the Protein Data Bank (PDB). The key constituents of basic amino acid residues on the surface of CHI and TLPs were lysine (Lys) and arginine (Arg), while acidic residues were mainly glutamic acid (Glu) and aspartic acid (Asp). These structural considerations support the hypothesis underpinning this study and suggest that basic amino acid residues along with the N-terminal may interact with negatively charged zeolite sites. Figure 2 shows a possible mechanism of adsorption for hazeforming proteins to natural zeolite. The natural zeolite
Figure 2. Schematic diagram of adsorption of the haze-forming protein onto natural zeolite.
structure has a net negative charge which facilitates trapping of positive ions such as potassium, calcium, magnesium, and sodium. These ions are held electronically within the open pores and are exchangeable with other positively charged entities.41,42 First, positively charged wine proteins approach the zeolite surface (Figure 2, left). In the next step, proteins are 12242
DOI: 10.1021/acssuschemeng.9b01583 ACS Sustainable Chem. Eng. 2019, 7, 12240−12247
Research Article
ACS Sustainable Chemistry & Engineering
Figure 3. Protein concentration in wine following treatment by different particle sizes of zeolite: (A) dry zeolites and (B) wet zeolites (in both cases dose of 4 g/L of zeolite was added to wines). Each point corresponds to the average of three repetitions.
Figure 4. Effect of hydration time and treatment time on protein concentration when using 2 g/L of zeolite (50−20 μm size fraction) to treat Sauvignon Blanc wine. Protein concentration was measured by HPLC. Each point corresponds to the average of three repetitions.
Figure 5. Effect of zeolite dose on the protein removal and turbidity of three different white wines.
and 70% in SEM, CHA, and SAB wines, respectively. Increases in zeolite dose resulted in further reductions in protein content in all three wines. At 6 g/L more than 90% of all protein was removed. In addition to the HPLC analysis, the heat test regularly used in the wine industry to test protein stability was also carried out. In this test, wines are considered stable when ΔNTU < 2 (ΔNTU = turb2 − turb1), where turb1 is the turbidity measured before and turb2 the turbidity measured after the heating step. Figure 5b shows that the Semillon wine was fully stabilized with 4 g/L of zeolite, whereas the Sauvignon Blanc and Chardonnay wines were fully stabilized at 6 g/L dosage of zeolite. Total protein content of the three wines, their turbidity, and the amount of two commonly used bentonites and zeolite required for stabilization are given in Table 2. This comparative analysis demonstrates that larger
Table 2. Protein Concentration and Turbidity of the Three Wines Useda
total protein content (mg/L) turbidity (NTU) Vitiben (g/L) Pluxcompact (g/L) natural zeolite (g/L)
Sauvignon Blanc
Semillon
Chardonnay
182 ± 5 26.7 ± 0.5 0.8 1.3 6
165 ± 3 30 ± 0.8 1.2 1.8 4
103 ± 2 29.5 ± 0.7 0.8 1 6
a
Amount of commonly used bentonites (Vitiben and Pluxcompact) and zeolite required for heat stabilization.
amounts of zeolite are required to achieve the same level of haze protein stabilization as the bentonites. However, the 12243
DOI: 10.1021/acssuschemeng.9b01583 ACS Sustainable Chem. Eng. 2019, 7, 12240−12247
Research Article
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a
Data from single determination except base wines where data represent the average of five independently prepared and tested samples. bCd, Cr, Co, Pb, Ni, and Se were not presented since their concentrations were below the limit of detection.
1075 64 106 38 0.7
