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Enzymatic process for the fractionation of baker’s yeast cell wall (Saccharomyces cerevisiae)
Food Chemistry 163 (2014) 108–113
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Enzymatic process for the fractionation of baker’s yeast cell wall (Saccharomyces cerevisiae) Chema Borchani a,⇑, Fabienne Fonteyn b, Guilhem Jamin b, Michel Paquot c, Christophe Blecker a, Philippe Thonart b a b c
Université de Liège, Gembloux Agro-Bio Tech, Unité de Science des Aliments et Formulation, Passage des Déportés 2, B-5030 Gembloux, Belgium Université de Liège, Gembloux Agro-Bio Tech, Unité de Bioindustries, Passage des Déportés 2, B-5030 Gembloux, Belgium Université de Liège, Gembloux Agro-Bio Tech, Unité de Chimie Analytique, Passage des Déportés 2, 5030 Gembloux, Belgium
a r t i c l e
i n f o
Article history: Received 22 October 2013 Received in revised form 10 March 2014 Accepted 23 April 2014 Available online 2 May 2014 Keywords: b-Glucans Baker’s yeast Saccharomyces cerevisiae Enzymatic process Yield Chemical properties
a b s t r a c t b-Glucans, homopolymers of glucose, are widespread in many microorganisms, mushrooms and plants. They have attracted attention because of their bioactive and medicinal functions. One important source of b-glucans is the cell wall of yeasts, especially that of baker’s yeast Saccharomyces cerevisiae. Several processes for the isolation of b-glucans, using alkali, acid or a combination of both, result in degradation of the polymeric chains. In this paper, we have an enzymatic process for the isolation of glucans from yeast cell walls. As a result, b-glucans were obtained in a yield of 18.0% of the original ratio in the yeast cell walls. Therefore, this isolation process gave a better yield and higher b-glucan content than did traditional isolation methods. Furthermore, results showed that each extraction step of b-glucan had a significant effects on its chemical properties. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The yeast cell wall consists mainly of polysaccharides, made up of glucose, mannose, and N-acetylglucosamine. One of the glucose polysaccharides, b-(1 ? 3) glucan, is the major structural component of the cell wall. Another b-(1 ? 6) glucan is relatively minor in amount but very important for cross linking (Cabib, Dong-Hyun, Schmidt, Crotti, & Varma, 2001). The mannose polysaccharides are linked to proteins to form a mannoprotein layer, mainly localized at the external surface. This mannoprotein retains the periplasmic proteins and limits the accessibility of foreign enzymes to the cell (De Nobel, Klis, Munnik, Priem, & Van Den Ende, 1990; Ziotnik, Fernandez, Bowers, & Cabib, 1984). Finally, a small amount of N-acetylglucosamine present in the wall (1–3%) is in the chitin (Cabib et al., 2001). Yeast is a unicellular fungus and has been used for baking and ethanol production for thousands of years. The worldwide production exceeded 2.5 millions tons in 2003. Some yeast factories made not only yeast for bakeries, but also produce yeast extracts, which are obtained after mechanically or enzymatically supported autolysis. The inner parts of the cells are isolated and used as food
⇑ Corresponding author. Tel./fax: +216 74 675 761. E-mail address: [email protected] (C. Borchani). http://dx.doi.org/10.1016/j.foodchem.2014.04.086 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
supplements and flavour enhancers due to their high amounts of proteins and nucleotides. The outer parts of the cell walls remain as waste, for which no commercial use has been established, except as a supplement for animal feed (Freimund, Sauter, Käppeli, & Dutler, 2003). First preparations from baker’s yeast yielded a glucan-enriched product, which revealed health-promoting properties and which contained approximately 55% of b-glucan, 19% mannan, 15% protein and 7% lipid (Freimund et al., 2003). b-Glucan is a glucose homopolymer compound of the cell wall of cereals, fungi and yeasts, distinguished by the type of glycosidic linkage between glycopyranose residues. An important source of this polysaccharide is the cell wall of yeasts, particularly of baker’s and brewer’s yeast, Saccharomyces cerevisiae (Dijkgraaf, Li, & Bussey, 2002). b-Glucan represents up to 20–60% of the cell dry weight (Freimund et al., 2003; Kim & Yun, 2006; Magnani et al., 2009). Several studies have shown that the glucan from S. cerevisiae is a very potent stimulator of the immune system. In fact, it protects the human body against viral, bacterial and fungal infections, tumors, radiation effects, and stress-related immuno-suppression (Bohn & BeMiller, 1995; Jaehrig et al., 2008; Magnani et al., 2009; Whistler, Bushway, Sighn, Nakahara, & Tokuzen, 1978). It also has potent antioxidant properties and free radical-scavenging capabilities. Moreover, b-glucan increases the effectiveness of
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antibiotics and reduces the LDL cholesterol level in the body (Kokashis, Williams, Cook, & Di Luzio, 1978; Robbins & Seeley, 1977). Especially, the b-glucan obtained from the yeast cell wall is more effective than is the b-glucan obtained from other sources (Kim & Yun, 2006). Indeed, several publications describe the strong positive influence of yeast b-glucan on the human and animal immune system (Bohn & BeMiller, 1995; Fitzpatrick, Haynes, Silver, & Dicarlo, 1964; Jaehrig et al., 2008 Li, Li, Xing, Cheng, & Lai, 2006). Owing to its health benefits, many processes for the isolation and purification of the b-glucan have been developed. Most of them usually employ acid, alkali or a combination of both, which contribute to degradation of glucosidic chains of the polymer, reduced yields and limited purity (Freimund et al., 2003; Magnani et al., 2009). In order to maintain the native structure of the glucan, research has revealed extraction with hot water and enzyme treatments as an alternative method for obtaining b-glucan from S. cerevisiae (Freimund et al., 2003; Jaehrig et al., 2008). Moreover, Liu, Wang, Cui, and Liu (2008) used an additional high-pressure homogenization step to aid in disrupting the yeast cell wall, and showed not only significant yields, but also preservation of the glucose chains. The present paper focusses on an enzymatic process for the fractionation of baker’s yeast cell wall (S. cerevisiae) via optimized steps involving hot water and enzymatic treatments (Savinase and Lipolase) in order to obtain the biologically active compounds of great interest. Thus, the chemical composition and the glucan content of all cell wall fractions (sediments and supernatants) were studied. 2. Materials and methods 2.1. Materials Yeast cell walls (S. cerevisiae) were provided by Puratos (Andenne, Belgium) and they were supplied as spray-dried powders. The protease (SavinaseÒ) and the lipase (LipolaseÒ) enzymes were provided by Sigma Aldrich (Belgium). All reagents used were of analytical grade. 2.2. Processing of the yeast cell walls 2.2.1. General Glucan was enriched in the yeast cell walls by a procedure of Jaehrig et al. (2008) with modifications. The schematic process for the fractionation is shown in Fig. 1.
Native YCW (F1) Extraction with H2O, pH 7, 125 °C, 5 h; centrifugation
Supernatant (S1): mannoproteins, soluble glucans
Sediment (F2): glucans, proteins, lipids Treatment with protease, pH 10.5, 45 °C, 5 h; centrifugation
Supernatant (S2): peptides
Sediment (F3): glucans, lipids Treatment with lipase, pH 10.5, 45 °C, 5 h; centrifugation
Supernatant (S3): glycerol, fatty acids
Sediment (F4): glucans
Fig. 1. Schematic process for the fractionation of yeast cell walls (YCW) adapted from Jaehrig et al. (2008).
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2.2.2. Hot water extraction A suspension of yeast cell wall (YCW) (F1) in distilled H2O (10% w/v) was adjusted to pH 7 with 9.98 M NaOH (30% w/v). The suspension was heated to 125 °C in a steam autoclave (MBG). After 5 h, the suspension was cooled and centrifuged. Thus, the insoluble residue obtained by centrifugation (10,000g, 2 °C, 15 min) (SORVALLÒ RC 12 BP, USA) was washed twice with distilled water. The sediment was lyophilised (F2) and the supernatant (S1) was stored at 20 °C. 2.2.3. Protease treatment Subsequent to hot water extraction, the washed sediment (fraction F2) was diluted with distilled H2O to 6.7% (w/v) dry weight and incubated in a fermenter with mild agitation. After heating to 45 °C and adjustment of the pH to 10.5 with 9.98 M NaOH (30% w/v), the protease SavinaseÒ (Sigma Aldrich) (0.17% v/v) was added under stirring at t = 0, 1.5 and 3 h. After an overall duration of 5 h, the insoluble residue was separated by centrifugation (10,000g, 2 °C, 15 min) (Essoreuse Rousselet RC 40 VXR) and washed twice with distilled water. The sediment (fraction F3) was lyophilised and the supernatant (S2) was stored at 20 °C. 2.2.4. Lipase treatment Following both hot water extraction (fraction F2) and protease treatment (fraction F3), the sediment was diluted with distilled water to 4% (w/v) dry weight and incubated in a fermenter with mild agitation. Further, the suspension was treated with LipolaseÒ (Sigma Aldrich) (0.10% v/v) at 45 °C and pH 10.5 with stirring for 3 h. The insoluble residue was separated by centrifugation (10,000g, 2 °C, 15 min) (SORVALLÒ RC 12 BP, USA) and washed twice with distilled water. The sediment (fraction F4) was lyophilised and the supernatant (S3) was stored at 20 °C. Thus, different fractions of yeast cell walls (lyophilised sediments and supernatants) were analysed. 2.3. Analytical methods 2.3.1. Measurement of dry matter and ash Moisture and ash contents of both sediments and supernatants of the cell wall fractions were determined according to the approved methods of the American Association of Cereal Chemists: Method 44-16 and Method 08-01, respectively (AACC, 2003). Dry matter was determined by drying 1 g of samples in a hot air oven at 120 °C to constant weights. Ash content was determined after incineration at 550 °C overnight, using a muffle furnace (NABER, Germany). The total ash was expressed as percent of dry weight. 2.3.2. Measurement of fat Fat content was determined according to the method described by Lecoq (1965), with petroleum ether as the organic solvent. Dried material (2 g) was suspended in 50 ml of 4 N HCl. The suspension was heated under reflux for 1 h. After cooling, the mixture was filtered. The residue was washed with petroleum ether. The combined filtrates were evaporated, yielding coloured oil, which was weighed. 2.3.3. Measurement of total nitrogen Nitrogen content, of both sediments and supernatants of the cell wall fractions, was determined using the Dumas combustion method (Rapid N Cube Elementar Analyser system GmbH, Germany). This involves a total combustion of the matrix under oxygen. The gases produced are reduced by copper and then dried, while the CO2 is trapped. The nitrogen is then quantified using a universal detector. Nitrogen content was reported as protein N 6.25.
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2.3.4. Analysis of macro- and micro-elements After ashing, the residue was dissolved in HNO3 and the mineral constituents (Ca, K, Na, Mg, Fe and Zn) were determined, using an atomic absorption spectrophotometer (Perkin-Elmer AAS 800, USA). Phosphorus content (P) was determined by the phosphomolybdovanadate method (AOAC, 1990). 2.3.5. Monosaccharide composition analysis Composition and quantity of monosaccharides (glucose, xylose, mannose, rhamnose arabinose and galactose) and the sum of monosaccharides were analysed with an HP 6890 gas chromatography system equipped with an HP-1 column (30 m 0.32 mm 0.25 lm). A FID detector heated at 320 °C and a constant flow (1.6 ml/min) of helium as the carrier gas were used. After injection, the temperature started at 120 °C for 1 min and was subsequently increased to 220 °C with a rate of 4 °C/ min. Finally, the temperature increased to 290 °C with a rate of 35 °C/min. Prior to the analysis, a dried sample (50 mg) was suspended in aqueous sulphuric acid (72%, 0.5 ml) and stirred at 30 °C for 1 h. The Individual neutral sugars were released from both sediments and supernatants of the cell wall fractions by acid hydrolysis with 1 M H2SO4 (5.5 ml of distilled water) for 6 h, autoclaving at 100 °C according to the modified method of Thygesen, Hansen, and Jacobsen (2005). Then, they were converted to alditol acetates by acetic anhydride in the presence of methylimidazole, after reduction by sodium tetraborate (Blakney, Harris, Henry, & Stone, 1983). Hydrolysis was done in duplicate and, after cooling, the monosaccharide solutions were neutralised with NaOH to a pH range of 5–7. A correction factor was used, based on the loss of pure monosaccharides during the acid hydrolysis. 2.3.6. b-Glucan content determination The b-glucan assay was conducted according to the method of McCleary and Glennie-Holmes (1985), using the b-glucan enzymatic assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland). b-Glucan was solubilised in concentrated hydrochloric acid (HCl) (37%, 10 N) and then extensively hydrolysed by 1.3 N HCl at 100 °C for 2 h. After that, 10 ml of 2 N KOH were added to the suspensions of samples and then the whole was transferred to a 100 ml volumetric flask, using 200 mM sodium acetate buffer (pH 5). The suspensions of samples were filtered through Whatman GF/A glass fibre filter paper before reading the colour. Hydrolysis to D-glucose was completed by incubation at 40 °C for 60 min with a mixture of highly purified exo-1,3-b-glucanase (20 U/ml) and b-glucosidase (4 U/ml). Finally, 3 ml of glucose oxidase/peroxidase mixture were added to each suspension and the mixture was incubated at 40 °C for 20 min. The measurements were carried out with a spectrophotometer (GENESYS 10 S uv–vis) and the absorbance of all solutions was measured at 510 nm against a blank which consisted of 0.2 ml of sodium acetate buffer (200 mM, pH
5) and 3 ml of glucose oxidase/peroxidase reagent. The b-glucan contents of both sediments and supernatants of the cell wall fractions were recorded on a moisture free basis. 2.4. Statistical analysis The analyses were performed in duplicate. The obtained data were analyzed using the ANOVA technique by the Statistical Package for the Social Sciences ‘‘SPSS’’ (version 11). Significance of differences between groups was evaluated using Duncan’s multiple range tests at the level of p < 0.01 (SPSS, 1997). 3. Results and discussion 3.1. Yields of fractions of the glucan isolation process This process, adapted from Jaehrig et al. (2008), starts with a hot water extraction in order to remove mannoproteins and a part of the soluble glucans. Subsequently, the treatments with enzymes, especially Savinase and Lipolase were used to remove the peptides and lipids (glycerol, fatty acids). Freimund et al. (2003) reported that enzymatic treatments have the benefit of preserving the native structure and of avoiding chain degradation of glucan. The step to step yields of both sediments F2, F3 and F4 and of supernatants S1, S2 and S3 of the cell wall fractions were 52.7%, 47.7%, 71.6%, 40.8%, 51.2% and 28.9% (w/w), respectively (Fig. 2). Compared with traditional isolation methods, our method had distinct advantages in that it gave a high yield of b-glucans and maintained the b-glucans’ native conformation. Furthermore, this isolation method could be easily scaled-up to an industrial process. In this study, the hot water extraction step of yeast cell wall resulted in a loss of weight, which can mainly be attributed to removal of protein and mannoprotein from the starting material. Additionally, our process gives a lower yield (expressed in weight of the starting material) (18.0% vs 25–26%) than that reported by Freimund et al. (2003). In fact, the 10% yield (lowest) could be explained by the hot water extraction step not being under agitation, like that of Liu et al. (2008). So, this step had a yield closer to ours as is also the case for the final yield (21.4% of yeast cell walls). Nevertheless, both of these reports combined a protease treatment with an organic solvent delipidation step. 3.2. Monosaccharide and total carbohydrate contents of fractions In order to determine the content of polysaccharides, especially to clarify the important question of the glucan purity, our analyses began with an acid hydrolysis. Sulphuric acid is the most used reagent for the cleavage of polysaccharides, producing the monosaccharides, which build up the polymer chains (Freimund et al.,
Fig. 2. Yields of fractions of the glucan isolation process. A native yeast cell wall (F1), B F2 and S1 after hot water treatment, C F3 and S2 after hot water and protease treatment, D F4 and S3 after hot water, protease and lipase treatment.
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C. Borchani et al. / Food Chemistry 163 (2014) 108–113 Table 1 Chemical composition (% dry weight basis) of fractions of the glucan isolation process. Fraction
Total carbohydrates a
F1 F2 F3 F4 S1 S2 S3
Protein
Lipid d
42.3 ± 2.80 42.3 ± 5.68a 59.7 ± 16.88a 81.1 ± 6.27a 31.9 ± 3.42B 18.9 ± 1.33AB 11.4 ± 4.17A
Ash a
27.6 ± 0.09 21.6 ± 0.09c 13.3 ± 0.19b 8.44 ± 0.18a 33.9 ± 0.07B 23.2 ± 0.31A 22.4 ± 0.38A
12.6 ± 0.08 19.9 ± 0.33c 15.3 ± 0.02b 14.9 ± 0.06b 1.30 ± 0.04C 7.96 ± 0.50A 4.76 ± 0.14B
Massa
Dry matter d
7.00 ± 0.02 6.17 ± 0.01c 5.73 ± 0.01b 2.94 ± 0.04a 14.0 ± 0.07A 20.6 ± 0.23B 35.2 ± 0.01C
a
94.4 ± 0.15 95.3 ± 0.11b 96.6 ± 0.01c 95.4 ± 0.06b 92.2 ± 0.05B 87.7 ± 0.33A 87.5 ± 0.08A
600.32 316.56 150.88 108 – – –
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment. a Expressed in g.
Table 2 Monosaccharides composition (% dry weight basis) of fractions of the glucan isolation process. Fraction
Rha
Ara
Xyl
Man
Glu
Gal
NI
NI
F1 F2 F3 F4 S1 S2 S3
0.29 ± 0.01a 0.09 ± 0.09a 0.08 ± 0.08a 0.00 ± 0.00a 0.12 ± 0.03A 0.10 ± 0.03A 0.88 ± 0.71A
0.01 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.07 ± 0.02A 0.02 ± 0.02A 0.45 ± 0.45A
0.01 ± 0.01a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.05 ± 0.02A 0.00 ± 0.00A 0.00 ± 0.00A
9.28 ± 1.17a 5.63 ± 2.08a 4.00 ± 1.83a 2.11 ± 0.36a 11.52 ± 0.66B 3.63 ± 0.24A 3.31 ± 0.18A
32.6 ± 1.61a 36.6 ± 3.50a 55.6 ± 15.0a 79.0 ± 5.91a 7.76 ± 0.54B 9.24 ± 0.24B 3.29 ± 0.28A
0.02 ± 0.01a 0.01 ± 0.01a 0.00 ± 0.00a 0.00 ± 0.00a 0.10 ± 0.03A 0.04 ± 0.03A 0.00 ± 0.00A
– – – – 3.09 ± 0.47A 3.19 ± 0.05A 2.17 ± 0.81A
– – – – 9.17 ± 0.65B 2.70 ± 0.33A 1.34 ± 0.52A
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. Rha rhamnose, Ara arabinose, Xyl xylose, Man mannose, Glu glucose, Gal galactose, NI non-identified. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment.
2003). Tables 1 and 2 show the composition of monosaccharides and the content of polysaccharides of both sediments and supernatants of the cell wall fractions. The increase in total carbohydrate content from fractions F1 to F4 (42.3–81.1g) is due to the structure of the cell wall and the extraction method used in this study. In fact, the hot water extraction step has been proven to be very efficient to remove proteins and soluble polysaccharides from yeast cell walls. The glucose content increased from fractions F1 to F4, which represent the b-glucan extract with a glucose content of 79.0% of dry weight basis (purity). Other authors (Freimund et al., 2003; Liu et al., 2008; Magnani et al., 2009) have reported glucose contents greater than 90%. The decrease in mannose content from fractions F1 to F4 (9.28–2.11) and from S1 to S3 (11.5–3.31) is explained by the hot water and enzyme treatments applied in this study. Freimund et al. (2003) reported that extraction of water-soluble compounds from biological materials with hot water is an approved procedure. This step was used to remove water-soluble components of the yeast cell walls, particularly mannoprotein and other proteins. So, the hot water extraction and protease steps seemed to work efficiently for removal of mannoproteins. In the hot water extraction step, we found a temperature of 125 °C for five hours to be the optimum conditions for removal of mannoproteins and other compounds. Moreover, the decrease of glucose and mannose is due to the acidic conditions, such as hydrochloric, sulfuric and trifluoroacetic acid. According to the literature, the most common procedure is treatment with acids for depolymerising glucan and mannan. Reaction time, temperature and acid concentration have to be chosen, under which conditions the polysaccharides are completely cleaved but which ensure a less degradation of the D-glucose and D-mannose released because they are sensitive
to acidic degradation at high temperatures (Freimund, Janett, Arrigoni, & Amadò, 2005). 3.3. Protein content The protein content of b-glucan may affect the various functional properties of products in which b-glucan is added as a functional ingredient. So the residual protein is a key parameter. Several authors have reported that the removal of a significant amount of proteins was due to hot water treatment and action of the protease enzyme (Ahmad, Anjum, Zahoor, Nawaz, & Din, 2009; Freimund et al., 2003; Jaehrig et al., 2008; Liu et al., 2008). In our study, protein contents varied significantly (p < 0.01) among the cell wall fractions and ranged from 8.44% to 27.6% (Table 1). The protein content of the supernatants S1, S2 and S3 were 33.9%, 23.2% and 22.4% on dry basis, respectively. The significant reduction of protein content from F2 to F3 is due to protease activity. A significant loss of protein was also observed during the lipase treatment, probably due to proteolytic coactivity, as noted by Jaehrig et al. (2008). Totally, about 95% of the proteins were released. This value is in agreement with results in the literature (Freimund et al., 2003). 3.4. Lipid content An elevated lipid content (12.6%) was observed in the yeast cell wall used. This would correspond to the presence of plasma membrane in the product (Dallies, François, & Paquet, 1998; Klis, 1994). This value was close to that reported by Freimund et al. (2003). Lipid contents varied substantially (p < 0.01) among F1, F2 and F3 but surprisingly there was no difference between F3 and F4 (Table 1). This means that the remaining lipids are not typical
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Table 3 Minerals contents (mg/kg dry weight basis) of fractions of the glucan isolation process. Fraction F1 F2 F3 F4 S1 S2 S3
P
K c
17804 ± 0.08 10448 ± 0.02b 5554 ± 0.00a 5295 ± 0.03a 1357 ± 24.2B 91.3 ± 4.19A 1468 ± 72.3B
Ca b
6035 ± 0.01 3144 ± 0.02ab 2768 ± 0.18ab 609 ± 0.00a 521 ± 3.76C 20.3 ± 0.08A 103 ± 2.02B
Na a
2030 ± 0.00 2954 ± 0.01c 2324 ± 0.00b 2299 ± 0.01b 8.78 ± 0.67A 11.7 ± 0.95A 8.17 ± 0.90A
Mg a
582 ± 0.00 9332 ± 0.02c 20274 ± 0.01d 7066 ± 0.01b 1474 ± 3.38C 217.06 ± 4.92A 1319 ± 4.84B
Zn c
837 ± 0.00 924 ± 0.00d 718 ± 0.00b 506 ± 0.00a 26.8 ± 0.11C 7.53 ± 0.07A 11.4 ± 0.09B
Cu a
271 ± 4.24 433 ± 8.49b 440 ± 1.41b 474 ± 1.41c 1.78 ± 0.05A 1.66 ± 0.06A 2.08 ± 0.03B
Fe a
5.62 ± 0.07 8.29 ± 0.28c 7.25 ± 0.07b 5.97 ± 0.07a 96.1 ± 18.5A 110 ± 0.33A 117 ± 1.77A
Mn a
47.78 ± 2.40 63.3 ± 4.10ab 64.1 ± 0.42ab 75.9 ± 8.06b 1.20 ± 0.18A 0.63 ± 0.04A 0.81 ± 0.01A
7.31 ± 0.21a 10.3 ± 0.28b 13.5 ± 0.35c 11.7 ± 0.78bc 0.11 ± 0.00A 0.18 ± 0.04A 0.23 ± 0.02A
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment.
Table 4 Total, a and b-glucan contents (% dry weight basis) of fractions of the glucan isolation process. Fraction
Total glucan
a-Glucan
b-Glucan
F1 F2 F3 F4 S1 S2 S3
15.6 ± 0.77a 32.2 ± 1.60b 41.5 ± 2.64c 54.4 ± 0.95d 11.12 ± 0.76B 20.5 ± 0.30C 4.98 ± 0.40A
2.17 ± 0.18b 2.21 ± 0.22b 1.03 ± 0.13a 0.99 ± 0.05a 5.25 ± 0.34C 3.08 ± 0.06B 0.62 ± 0.11A
13.4 ± 0.59a 30.0 ± 1.38b 40.5 ± 2.76c 53.4 ± 1.00d 5.90 ± 1.10A 17.5 ± 0.36B 4.36 ± 0.51A
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment.
fats. In fact, it is known that, besides glycerides, several sterols and squalene are components of the lipid fraction in yeasts (Peña & Sandra, 1995). Blagovic´, Rupcˇic´, Mesaric´, Georgiú, and Maric´ (2001) found that squalene constituted 33% of total lipids in a brewer’s yeast. This might explain the apparent lack of lipase activity in our study. So, Magnani et al. (2009) reported that most of the b-D-glucan extraction processes do not include lipid extraction before enzyme treatment, since they may interfere with the protease action. Several organic solvents have been used for extraction of lipids during the removal of the b-D-glucan from S. cerevisiae. Liu et al. (2008) investigated extraction under reflux with isopropanol; however, Freimund et al. (2003) extracted only part of the lipids with the same solvent. Compared to our study, these extraction processes give low values of lipids (or traces).
3.5. Ash content and mineral composition Significant differences (p < 0.01) in ash contents were observed between fractions of the glucan isolation process. The ash content of b-glucan (2.94%) from baker’s yeast (S. cerevisiae) was higher than that of b-glucan from other sources, such as barley and oat cereals (1.42%, 1.56%; respectively) (Ahmad, Anjum, Zahoor, Nawaz, & Ahmed, 2010; Ahmad et al., 2009). Additionally, the significant amount of ash observed in supernatants might be due to process steps. This result is explained by increase in the solubility of minerals due to the number of washing proposed in this study.
Dietary fibre has long been known to form chelates with a number of divalent metals. These minerals have a capacity to modify functional properties of dietary fibre and offer a buffering action in solutions (Ahmad et al., 2010). The amounts of macro- and micro-elements detected in both supernatants and sediments of the cell wall fraction are shown in Table 3. The sediment of fraction F1 contains appreciable amounts of phosphorus, potassium and calcium. Moderate amounts of magnesium, sodium and zinc were also observed (837, 582 and 271 mg/kg dry weight basis). Minor amounts of iron, manganese and copper were also observed. In the present study, sodium, phosphorus and calcium were the dominating mineral components of extracted b-glucan (fraction F4). The amounts of zinc and iron in fraction F4 were 474 and 75.9 mg/kg dry weight basis. Contents of zinc and iron were approximately two times higher in the purified b-glucan powder than in the dry matter of the yeast cell wall extracts. Indeed, the abundance of iron (in higher concentrations in purified b-glucan sample) suggests that iron was associated with b-glucan rather than with phytate or proteins (Kivelä, Sontag-Strohm, Loponen, Tuomainen, & Nyström, 2011). The supernatant fractions S1 and S3 contained significant amount of minerals (Table 3). Phosphorus and sodium concentrations were the highest (1357–1468 mg/kg dry mass and 1319–1474 mg/kg dry mass, respectively) and modulate pH, followed in descending order by potassium (103– 521 mg/kg dry mass), copper (96.1–117 mg/kg dry mass), magnesium (11.4–26.8 mg/kg dry mass), calcium (8.17–8.78 mg/kg dry mass), zinc (1.78–2.08 mg/kg dry mass), iron (0.81–1.20 mg/kg dry mass) and manganese (0.11–0.23 mg/kg dry mass). Besides, the fraction S2 had only tiny amounts of P, K, Ca, and Na compared with other supernatant fractions (Table 3). Generally, 30% of sugars in S2 and S3 were not identified. This might be explained by the chosen conditions of acid hydrolysis. 3.6. Total, a- and b-glucan contents The content of total, a- and b-glucan of both sediment and supernatant of the cell wall fractions are shown in Table 4. The fraction with the lowest amount of glucan was the F1 fraction, which represents the native yeast cell wall. The highest glucan content (54.4%) was in the fraction F4 when hot water, protease and lipase treatments were applied for extraction of b-glucan. The significant increase in glucan content from fractions F1 to F4 is due to the extraction and the purification steps of b-glucan. Thus, our process steps gave better yield and higher purity than the traditional isolation process involving treatments with hot alkali and acids. The supernatant fraction with the highest amount of glucan was the S2 fraction. Statistical analyses show significant differences (p < 0.01) in total glucan content among both sediments and supernatants of the cell wall fractions. Actually, these differ-
C. Borchani et al. / Food Chemistry 163 (2014) 108–113
ences could be explained by the way the enzyme works. In the other words, the extraction process used in our study showed a significant difference in total glucan content due to the extraction steps comprising the hot water and the Savinase and Lipolase enzyme activities. 4. Conclusions In the present paper, an enzymatic isolation process adapted from Jaehrig et al. (2008), has been established for producing valuable b-glucan from baker’s yeast. Our process has some distinct advantages compared to previously published isolation processes using the solvent treatment. First, the soft treatments with water and enzymes avoid chain degradation and therefore preserve the original glucan structure of the cell wall. Second, b-glucan was obtained in a yield of 18.0% (w/w) of the original ratio in the yeast cell walls and a purity of 79.0%. Finally, the choice of yeast isolation process appeared to be important as it affected most of the chemical properties e.g. mineral and total glucan contents among the sediment and the supernatant fractions. Enzymatic treatment steps with Savinase and Lipolase enzymes were found to be good since they gave high b-glucan content, but also removed more ash, fat and protein during the extraction of b-glucan. In conclusion, the isolation process used in this study is inexpensive and it is easy to produce b-glucan from S. cerevisiae for industrial utilisation. References AACC (2003). Approved methods of American Association of Cereal Chemists. St Paul: The American Association of Cereal Chemists Inc.. Ahmad, A., Anjum, F. M., Zahoor, T., Nawaz, H., & Ahmed, Z. (2010). Extraction and characterization of b-D-glucan from oat for industrial utilization. International Journal of Biological Macromolecules, 46, 304–309. Ahmad, A., Anjum, F. M., Zahoor, T., Nawaz, H., & Din, A. (2009). Physicochemical and functional properties of barley b-glucan as affected by different extraction procedures. International Journal of Food Science & Technology, 44, 181–187. AOAC (1990). Official methods of analysis (15th ed.). Arlington: The Association of the Official Analytical Chemists. Blagovic´, B., Rupcˇic´, J., Mesaric´, M., Georgiú, K., & Maric´, V. (2001). Lipid composition of brewer’s yeast. Food Technology and Biotechnology, 39, 175–181. Blakney, A. B., Harris, P. J., Henry, R. J., & Stone, B. A. (1983). A simple and rapid preparation of aldithol acetates for monosaccharide analysis. Carbohydrate Research, 113, 291–299. Bohn, J. A., & BeMiller, J. N. (1995). (1 ? 3)-b-D-Glucans as biological response modifiers: A review of structure-functional activity relationships. Carbohydrate Polymers, 28, 3–14. Cabib, E., Dong-Hyun, R., Schmidt, M., Crotti, L. B., & Varma, A. (2001). The yeast cell wall and septum as paradigms of cell growth and morphogenesis. Journal of Biological Chemistry, 276, 19679–19682. Dallies, N., François, J., & Paquet, V. (1998). A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast, 14, 1297–1306.
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De Nobel, J. G., Klis, F. M., Munnik, T., Priem, J., & Van Den Ende, H. (1990). An essay of relative cell wall porosity in Saccharomyces cerevisiae, Kluyveromyces lactis, and Schizosacchromyces pombe. Yeast, 6, 483–490. Dijkgraaf, G. J. P., Li, H., & Bussey, H. (2002). Cell-b-glucans of Saccharomyces cerevisiae. In E. J. Vandamme, S. De Baets, & A. Steinbüchel (Eds.), Biopolymers. Polysaccharides 2 (Vol. 6, pp. 79–213). Weinheim, Germany: Wiley-VCH Verlag. Fitzpatrick, F. W., Haynes, L. J., Silver, N. J., & Dicarlo, F. J. (1964). Effect of glucan derivatives upon phagocytosis by mice. Journal of the Reticuloendothelial Society, 15, 423–428. Freimund, S., Janett, S., Arrigoni, E., & Amadò, R. (2005). Optimised quantification method for yeast-derived 1,3-b-D-glucan and a-D-mannan. European Food Research and Technology, 220, 101–105. Freimund, S., Sauter, M., Käppeli, O., & Dutler, H. (2003). A new non-degrading isolation process for 1,3-b-D-glucan of high purity from baker’s yeast Saccharomyces cerevisiae. Carbohydrate Polymers, 54, 159–171. Jaehrig, S. C., Rohn, S., Kroh, L. W., Wildenauer, F. X., Lisdat, F., Fleischer, L.-G., & Kurz, T. (2008). Antioxidative activity of (1 ? 3), (1 ? 6)-b-D-glucan from Saccharomyces cerevisiae grown on different media. LWT-Food Science and Technology, 41, 868–877. Kim, K. S., & Yun, H. S. (2006). Production of soluble b-glucan from the cell wall of Saccharomyces cerevisiae. Enzyme and Microbial Technology, 39, 496–500. Kivelä, R., Sontag-Strohm, T., Loponen, J., Tuomainen, P., & Nyström, L. (2011). Oxidative and radical mediated cleavage of b-glucan in thermal treatments. Carbohydrate Polymers, 85(3), 645–652. Klis, F. M. (1994). Review: Cell wall assembly in yeast. Yeast, 10, 851–869. Kokashis, P. L., Williams, D. L., Cook, J. A., & Di Luzio, N. R. (1978). Increased resistance to Staphylococcus aureus infection and enhancement in serum activity by glucan. Science, 199, 1340–1342. Lecoq, R. (1965). Manuel d’analyse alimentaire et d’expertises usuelles. Tome 1. Edition Doin et Cie. Wiseman, p: 200–203, 569, 1604–1613. Li, J., Li, D. F., Xing, J. J., Cheng, Z. B., & Lai, C. H. (2006). Effects of b-glucan extracted from Saccharomyces cerevisiae on growth performance, and immunological and somatotropic responses of pigs challenged with Escherichia coli lipopolysaccharide. Journal of Animal Science, 84, 2374–2381. Liu, X. Y., Wang, Q., Cui, S. W., & Liu, H. Z. (2008). A new isolation method of b-Dglucans from spent yeast Saccharomyces cerevisiae. Food Hydrocolloids, 22, 239–247. Magnani, M., Calliari, C. M., de Macedo, F. C., Jr., Mori, M. P., de Syllos Cólus, I. M., & Castro-Gomez, R. J. H. (2009). Optimized methodology for extraction of (1 ? 3) (1 ? 6)-â-D-glucan from Saccharomyces cerevisiae and in vitro evaluation of the cytotoxicity and genotoxicity of the corresponding carboxymethyl derivative. Carbohydrate Polymers, 78, 658–665. McCleary, B. V., & Glennie-Holmes, M. (1985). Enzymic quantification of (1 ? 3), (1 ? 4) b-glucan in barley and malt. Journal of the Institute of Brewing, 91, 285–295. Peña, A., & Sandra, P. (1995). Chemotaxonomic characterization of yeast cells. Journal of Chromatographic Science, 33, 116–122. Robbins, E. A., & Seeley, R. D. (1977). Cholesterol lowering effect of dietary yeast and yeast fractions. Journal of Food Science, 42, 694–698. SPSS (1997). Guía del usuario del Sistema base de SPSS 7.5 para Windows. Chicago. Thygesen, K. S., Hansen, L. B., & Jacobsen, K. W. (2005). Partly occupied wannier functions. Physical Review Letters. http://dx.doi.org/10.1103/PhysRevLett. 94.026405. Whistler, R. L., Bushway, A., Sighn, P. P., Nakahara, W., & Tokuzen, R. (1978). Noncytotoxic, antitumor polysaccharides. Advances in Carbohydrate Chemistry and Biochemistry, 32, 235–275. Ziotnik, H., Fernandez, M. P., Bowers, B., & Cabib, E. (1984). Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity. Journal of Bacteriology, 159, 1018–1026.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Enzymatic process for the fractionation of baker’s yeast cell wall (Saccharomyces cerevisiae) Chema Borchani a,⇑, Fabienne Fonteyn b, Guilhem Jamin b, Michel Paquot c, Christophe Blecker a, Philippe Thonart b a b c
Université de Liège, Gembloux Agro-Bio Tech, Unité de Science des Aliments et Formulation, Passage des Déportés 2, B-5030 Gembloux, Belgium Université de Liège, Gembloux Agro-Bio Tech, Unité de Bioindustries, Passage des Déportés 2, B-5030 Gembloux, Belgium Université de Liège, Gembloux Agro-Bio Tech, Unité de Chimie Analytique, Passage des Déportés 2, 5030 Gembloux, Belgium
a r t i c l e
i n f o
Article history: Received 22 October 2013 Received in revised form 10 March 2014 Accepted 23 April 2014 Available online 2 May 2014 Keywords: b-Glucans Baker’s yeast Saccharomyces cerevisiae Enzymatic process Yield Chemical properties
a b s t r a c t b-Glucans, homopolymers of glucose, are widespread in many microorganisms, mushrooms and plants. They have attracted attention because of their bioactive and medicinal functions. One important source of b-glucans is the cell wall of yeasts, especially that of baker’s yeast Saccharomyces cerevisiae. Several processes for the isolation of b-glucans, using alkali, acid or a combination of both, result in degradation of the polymeric chains. In this paper, we have an enzymatic process for the isolation of glucans from yeast cell walls. As a result, b-glucans were obtained in a yield of 18.0% of the original ratio in the yeast cell walls. Therefore, this isolation process gave a better yield and higher b-glucan content than did traditional isolation methods. Furthermore, results showed that each extraction step of b-glucan had a significant effects on its chemical properties. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The yeast cell wall consists mainly of polysaccharides, made up of glucose, mannose, and N-acetylglucosamine. One of the glucose polysaccharides, b-(1 ? 3) glucan, is the major structural component of the cell wall. Another b-(1 ? 6) glucan is relatively minor in amount but very important for cross linking (Cabib, Dong-Hyun, Schmidt, Crotti, & Varma, 2001). The mannose polysaccharides are linked to proteins to form a mannoprotein layer, mainly localized at the external surface. This mannoprotein retains the periplasmic proteins and limits the accessibility of foreign enzymes to the cell (De Nobel, Klis, Munnik, Priem, & Van Den Ende, 1990; Ziotnik, Fernandez, Bowers, & Cabib, 1984). Finally, a small amount of N-acetylglucosamine present in the wall (1–3%) is in the chitin (Cabib et al., 2001). Yeast is a unicellular fungus and has been used for baking and ethanol production for thousands of years. The worldwide production exceeded 2.5 millions tons in 2003. Some yeast factories made not only yeast for bakeries, but also produce yeast extracts, which are obtained after mechanically or enzymatically supported autolysis. The inner parts of the cells are isolated and used as food
⇑ Corresponding author. Tel./fax: +216 74 675 761. E-mail address: [email protected] (C. Borchani). http://dx.doi.org/10.1016/j.foodchem.2014.04.086 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
supplements and flavour enhancers due to their high amounts of proteins and nucleotides. The outer parts of the cell walls remain as waste, for which no commercial use has been established, except as a supplement for animal feed (Freimund, Sauter, Käppeli, & Dutler, 2003). First preparations from baker’s yeast yielded a glucan-enriched product, which revealed health-promoting properties and which contained approximately 55% of b-glucan, 19% mannan, 15% protein and 7% lipid (Freimund et al., 2003). b-Glucan is a glucose homopolymer compound of the cell wall of cereals, fungi and yeasts, distinguished by the type of glycosidic linkage between glycopyranose residues. An important source of this polysaccharide is the cell wall of yeasts, particularly of baker’s and brewer’s yeast, Saccharomyces cerevisiae (Dijkgraaf, Li, & Bussey, 2002). b-Glucan represents up to 20–60% of the cell dry weight (Freimund et al., 2003; Kim & Yun, 2006; Magnani et al., 2009). Several studies have shown that the glucan from S. cerevisiae is a very potent stimulator of the immune system. In fact, it protects the human body against viral, bacterial and fungal infections, tumors, radiation effects, and stress-related immuno-suppression (Bohn & BeMiller, 1995; Jaehrig et al., 2008; Magnani et al., 2009; Whistler, Bushway, Sighn, Nakahara, & Tokuzen, 1978). It also has potent antioxidant properties and free radical-scavenging capabilities. Moreover, b-glucan increases the effectiveness of
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antibiotics and reduces the LDL cholesterol level in the body (Kokashis, Williams, Cook, & Di Luzio, 1978; Robbins & Seeley, 1977). Especially, the b-glucan obtained from the yeast cell wall is more effective than is the b-glucan obtained from other sources (Kim & Yun, 2006). Indeed, several publications describe the strong positive influence of yeast b-glucan on the human and animal immune system (Bohn & BeMiller, 1995; Fitzpatrick, Haynes, Silver, & Dicarlo, 1964; Jaehrig et al., 2008 Li, Li, Xing, Cheng, & Lai, 2006). Owing to its health benefits, many processes for the isolation and purification of the b-glucan have been developed. Most of them usually employ acid, alkali or a combination of both, which contribute to degradation of glucosidic chains of the polymer, reduced yields and limited purity (Freimund et al., 2003; Magnani et al., 2009). In order to maintain the native structure of the glucan, research has revealed extraction with hot water and enzyme treatments as an alternative method for obtaining b-glucan from S. cerevisiae (Freimund et al., 2003; Jaehrig et al., 2008). Moreover, Liu, Wang, Cui, and Liu (2008) used an additional high-pressure homogenization step to aid in disrupting the yeast cell wall, and showed not only significant yields, but also preservation of the glucose chains. The present paper focusses on an enzymatic process for the fractionation of baker’s yeast cell wall (S. cerevisiae) via optimized steps involving hot water and enzymatic treatments (Savinase and Lipolase) in order to obtain the biologically active compounds of great interest. Thus, the chemical composition and the glucan content of all cell wall fractions (sediments and supernatants) were studied. 2. Materials and methods 2.1. Materials Yeast cell walls (S. cerevisiae) were provided by Puratos (Andenne, Belgium) and they were supplied as spray-dried powders. The protease (SavinaseÒ) and the lipase (LipolaseÒ) enzymes were provided by Sigma Aldrich (Belgium). All reagents used were of analytical grade. 2.2. Processing of the yeast cell walls 2.2.1. General Glucan was enriched in the yeast cell walls by a procedure of Jaehrig et al. (2008) with modifications. The schematic process for the fractionation is shown in Fig. 1.
Native YCW (F1) Extraction with H2O, pH 7, 125 °C, 5 h; centrifugation
Supernatant (S1): mannoproteins, soluble glucans
Sediment (F2): glucans, proteins, lipids Treatment with protease, pH 10.5, 45 °C, 5 h; centrifugation
Supernatant (S2): peptides
Sediment (F3): glucans, lipids Treatment with lipase, pH 10.5, 45 °C, 5 h; centrifugation
Supernatant (S3): glycerol, fatty acids
Sediment (F4): glucans
Fig. 1. Schematic process for the fractionation of yeast cell walls (YCW) adapted from Jaehrig et al. (2008).
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2.2.2. Hot water extraction A suspension of yeast cell wall (YCW) (F1) in distilled H2O (10% w/v) was adjusted to pH 7 with 9.98 M NaOH (30% w/v). The suspension was heated to 125 °C in a steam autoclave (MBG). After 5 h, the suspension was cooled and centrifuged. Thus, the insoluble residue obtained by centrifugation (10,000g, 2 °C, 15 min) (SORVALLÒ RC 12 BP, USA) was washed twice with distilled water. The sediment was lyophilised (F2) and the supernatant (S1) was stored at 20 °C. 2.2.3. Protease treatment Subsequent to hot water extraction, the washed sediment (fraction F2) was diluted with distilled H2O to 6.7% (w/v) dry weight and incubated in a fermenter with mild agitation. After heating to 45 °C and adjustment of the pH to 10.5 with 9.98 M NaOH (30% w/v), the protease SavinaseÒ (Sigma Aldrich) (0.17% v/v) was added under stirring at t = 0, 1.5 and 3 h. After an overall duration of 5 h, the insoluble residue was separated by centrifugation (10,000g, 2 °C, 15 min) (Essoreuse Rousselet RC 40 VXR) and washed twice with distilled water. The sediment (fraction F3) was lyophilised and the supernatant (S2) was stored at 20 °C. 2.2.4. Lipase treatment Following both hot water extraction (fraction F2) and protease treatment (fraction F3), the sediment was diluted with distilled water to 4% (w/v) dry weight and incubated in a fermenter with mild agitation. Further, the suspension was treated with LipolaseÒ (Sigma Aldrich) (0.10% v/v) at 45 °C and pH 10.5 with stirring for 3 h. The insoluble residue was separated by centrifugation (10,000g, 2 °C, 15 min) (SORVALLÒ RC 12 BP, USA) and washed twice with distilled water. The sediment (fraction F4) was lyophilised and the supernatant (S3) was stored at 20 °C. Thus, different fractions of yeast cell walls (lyophilised sediments and supernatants) were analysed. 2.3. Analytical methods 2.3.1. Measurement of dry matter and ash Moisture and ash contents of both sediments and supernatants of the cell wall fractions were determined according to the approved methods of the American Association of Cereal Chemists: Method 44-16 and Method 08-01, respectively (AACC, 2003). Dry matter was determined by drying 1 g of samples in a hot air oven at 120 °C to constant weights. Ash content was determined after incineration at 550 °C overnight, using a muffle furnace (NABER, Germany). The total ash was expressed as percent of dry weight. 2.3.2. Measurement of fat Fat content was determined according to the method described by Lecoq (1965), with petroleum ether as the organic solvent. Dried material (2 g) was suspended in 50 ml of 4 N HCl. The suspension was heated under reflux for 1 h. After cooling, the mixture was filtered. The residue was washed with petroleum ether. The combined filtrates were evaporated, yielding coloured oil, which was weighed. 2.3.3. Measurement of total nitrogen Nitrogen content, of both sediments and supernatants of the cell wall fractions, was determined using the Dumas combustion method (Rapid N Cube Elementar Analyser system GmbH, Germany). This involves a total combustion of the matrix under oxygen. The gases produced are reduced by copper and then dried, while the CO2 is trapped. The nitrogen is then quantified using a universal detector. Nitrogen content was reported as protein N 6.25.
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2.3.4. Analysis of macro- and micro-elements After ashing, the residue was dissolved in HNO3 and the mineral constituents (Ca, K, Na, Mg, Fe and Zn) were determined, using an atomic absorption spectrophotometer (Perkin-Elmer AAS 800, USA). Phosphorus content (P) was determined by the phosphomolybdovanadate method (AOAC, 1990). 2.3.5. Monosaccharide composition analysis Composition and quantity of monosaccharides (glucose, xylose, mannose, rhamnose arabinose and galactose) and the sum of monosaccharides were analysed with an HP 6890 gas chromatography system equipped with an HP-1 column (30 m 0.32 mm 0.25 lm). A FID detector heated at 320 °C and a constant flow (1.6 ml/min) of helium as the carrier gas were used. After injection, the temperature started at 120 °C for 1 min and was subsequently increased to 220 °C with a rate of 4 °C/ min. Finally, the temperature increased to 290 °C with a rate of 35 °C/min. Prior to the analysis, a dried sample (50 mg) was suspended in aqueous sulphuric acid (72%, 0.5 ml) and stirred at 30 °C for 1 h. The Individual neutral sugars were released from both sediments and supernatants of the cell wall fractions by acid hydrolysis with 1 M H2SO4 (5.5 ml of distilled water) for 6 h, autoclaving at 100 °C according to the modified method of Thygesen, Hansen, and Jacobsen (2005). Then, they were converted to alditol acetates by acetic anhydride in the presence of methylimidazole, after reduction by sodium tetraborate (Blakney, Harris, Henry, & Stone, 1983). Hydrolysis was done in duplicate and, after cooling, the monosaccharide solutions were neutralised with NaOH to a pH range of 5–7. A correction factor was used, based on the loss of pure monosaccharides during the acid hydrolysis. 2.3.6. b-Glucan content determination The b-glucan assay was conducted according to the method of McCleary and Glennie-Holmes (1985), using the b-glucan enzymatic assay kit (Megazyme International Ireland Ltd., Wicklow, Ireland). b-Glucan was solubilised in concentrated hydrochloric acid (HCl) (37%, 10 N) and then extensively hydrolysed by 1.3 N HCl at 100 °C for 2 h. After that, 10 ml of 2 N KOH were added to the suspensions of samples and then the whole was transferred to a 100 ml volumetric flask, using 200 mM sodium acetate buffer (pH 5). The suspensions of samples were filtered through Whatman GF/A glass fibre filter paper before reading the colour. Hydrolysis to D-glucose was completed by incubation at 40 °C for 60 min with a mixture of highly purified exo-1,3-b-glucanase (20 U/ml) and b-glucosidase (4 U/ml). Finally, 3 ml of glucose oxidase/peroxidase mixture were added to each suspension and the mixture was incubated at 40 °C for 20 min. The measurements were carried out with a spectrophotometer (GENESYS 10 S uv–vis) and the absorbance of all solutions was measured at 510 nm against a blank which consisted of 0.2 ml of sodium acetate buffer (200 mM, pH
5) and 3 ml of glucose oxidase/peroxidase reagent. The b-glucan contents of both sediments and supernatants of the cell wall fractions were recorded on a moisture free basis. 2.4. Statistical analysis The analyses were performed in duplicate. The obtained data were analyzed using the ANOVA technique by the Statistical Package for the Social Sciences ‘‘SPSS’’ (version 11). Significance of differences between groups was evaluated using Duncan’s multiple range tests at the level of p < 0.01 (SPSS, 1997). 3. Results and discussion 3.1. Yields of fractions of the glucan isolation process This process, adapted from Jaehrig et al. (2008), starts with a hot water extraction in order to remove mannoproteins and a part of the soluble glucans. Subsequently, the treatments with enzymes, especially Savinase and Lipolase were used to remove the peptides and lipids (glycerol, fatty acids). Freimund et al. (2003) reported that enzymatic treatments have the benefit of preserving the native structure and of avoiding chain degradation of glucan. The step to step yields of both sediments F2, F3 and F4 and of supernatants S1, S2 and S3 of the cell wall fractions were 52.7%, 47.7%, 71.6%, 40.8%, 51.2% and 28.9% (w/w), respectively (Fig. 2). Compared with traditional isolation methods, our method had distinct advantages in that it gave a high yield of b-glucans and maintained the b-glucans’ native conformation. Furthermore, this isolation method could be easily scaled-up to an industrial process. In this study, the hot water extraction step of yeast cell wall resulted in a loss of weight, which can mainly be attributed to removal of protein and mannoprotein from the starting material. Additionally, our process gives a lower yield (expressed in weight of the starting material) (18.0% vs 25–26%) than that reported by Freimund et al. (2003). In fact, the 10% yield (lowest) could be explained by the hot water extraction step not being under agitation, like that of Liu et al. (2008). So, this step had a yield closer to ours as is also the case for the final yield (21.4% of yeast cell walls). Nevertheless, both of these reports combined a protease treatment with an organic solvent delipidation step. 3.2. Monosaccharide and total carbohydrate contents of fractions In order to determine the content of polysaccharides, especially to clarify the important question of the glucan purity, our analyses began with an acid hydrolysis. Sulphuric acid is the most used reagent for the cleavage of polysaccharides, producing the monosaccharides, which build up the polymer chains (Freimund et al.,
Fig. 2. Yields of fractions of the glucan isolation process. A native yeast cell wall (F1), B F2 and S1 after hot water treatment, C F3 and S2 after hot water and protease treatment, D F4 and S3 after hot water, protease and lipase treatment.
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C. Borchani et al. / Food Chemistry 163 (2014) 108–113 Table 1 Chemical composition (% dry weight basis) of fractions of the glucan isolation process. Fraction
Total carbohydrates a
F1 F2 F3 F4 S1 S2 S3
Protein
Lipid d
42.3 ± 2.80 42.3 ± 5.68a 59.7 ± 16.88a 81.1 ± 6.27a 31.9 ± 3.42B 18.9 ± 1.33AB 11.4 ± 4.17A
Ash a
27.6 ± 0.09 21.6 ± 0.09c 13.3 ± 0.19b 8.44 ± 0.18a 33.9 ± 0.07B 23.2 ± 0.31A 22.4 ± 0.38A
12.6 ± 0.08 19.9 ± 0.33c 15.3 ± 0.02b 14.9 ± 0.06b 1.30 ± 0.04C 7.96 ± 0.50A 4.76 ± 0.14B
Massa
Dry matter d
7.00 ± 0.02 6.17 ± 0.01c 5.73 ± 0.01b 2.94 ± 0.04a 14.0 ± 0.07A 20.6 ± 0.23B 35.2 ± 0.01C
a
94.4 ± 0.15 95.3 ± 0.11b 96.6 ± 0.01c 95.4 ± 0.06b 92.2 ± 0.05B 87.7 ± 0.33A 87.5 ± 0.08A
600.32 316.56 150.88 108 – – –
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment. a Expressed in g.
Table 2 Monosaccharides composition (% dry weight basis) of fractions of the glucan isolation process. Fraction
Rha
Ara
Xyl
Man
Glu
Gal
NI
NI
F1 F2 F3 F4 S1 S2 S3
0.29 ± 0.01a 0.09 ± 0.09a 0.08 ± 0.08a 0.00 ± 0.00a 0.12 ± 0.03A 0.10 ± 0.03A 0.88 ± 0.71A
0.01 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.07 ± 0.02A 0.02 ± 0.02A 0.45 ± 0.45A
0.01 ± 0.01a 0.00 ± 0.00a 0.00 ± 0.00a 0.00 ± 0.00a 0.05 ± 0.02A 0.00 ± 0.00A 0.00 ± 0.00A
9.28 ± 1.17a 5.63 ± 2.08a 4.00 ± 1.83a 2.11 ± 0.36a 11.52 ± 0.66B 3.63 ± 0.24A 3.31 ± 0.18A
32.6 ± 1.61a 36.6 ± 3.50a 55.6 ± 15.0a 79.0 ± 5.91a 7.76 ± 0.54B 9.24 ± 0.24B 3.29 ± 0.28A
0.02 ± 0.01a 0.01 ± 0.01a 0.00 ± 0.00a 0.00 ± 0.00a 0.10 ± 0.03A 0.04 ± 0.03A 0.00 ± 0.00A
– – – – 3.09 ± 0.47A 3.19 ± 0.05A 2.17 ± 0.81A
– – – – 9.17 ± 0.65B 2.70 ± 0.33A 1.34 ± 0.52A
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. Rha rhamnose, Ara arabinose, Xyl xylose, Man mannose, Glu glucose, Gal galactose, NI non-identified. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment.
2003). Tables 1 and 2 show the composition of monosaccharides and the content of polysaccharides of both sediments and supernatants of the cell wall fractions. The increase in total carbohydrate content from fractions F1 to F4 (42.3–81.1g) is due to the structure of the cell wall and the extraction method used in this study. In fact, the hot water extraction step has been proven to be very efficient to remove proteins and soluble polysaccharides from yeast cell walls. The glucose content increased from fractions F1 to F4, which represent the b-glucan extract with a glucose content of 79.0% of dry weight basis (purity). Other authors (Freimund et al., 2003; Liu et al., 2008; Magnani et al., 2009) have reported glucose contents greater than 90%. The decrease in mannose content from fractions F1 to F4 (9.28–2.11) and from S1 to S3 (11.5–3.31) is explained by the hot water and enzyme treatments applied in this study. Freimund et al. (2003) reported that extraction of water-soluble compounds from biological materials with hot water is an approved procedure. This step was used to remove water-soluble components of the yeast cell walls, particularly mannoprotein and other proteins. So, the hot water extraction and protease steps seemed to work efficiently for removal of mannoproteins. In the hot water extraction step, we found a temperature of 125 °C for five hours to be the optimum conditions for removal of mannoproteins and other compounds. Moreover, the decrease of glucose and mannose is due to the acidic conditions, such as hydrochloric, sulfuric and trifluoroacetic acid. According to the literature, the most common procedure is treatment with acids for depolymerising glucan and mannan. Reaction time, temperature and acid concentration have to be chosen, under which conditions the polysaccharides are completely cleaved but which ensure a less degradation of the D-glucose and D-mannose released because they are sensitive
to acidic degradation at high temperatures (Freimund, Janett, Arrigoni, & Amadò, 2005). 3.3. Protein content The protein content of b-glucan may affect the various functional properties of products in which b-glucan is added as a functional ingredient. So the residual protein is a key parameter. Several authors have reported that the removal of a significant amount of proteins was due to hot water treatment and action of the protease enzyme (Ahmad, Anjum, Zahoor, Nawaz, & Din, 2009; Freimund et al., 2003; Jaehrig et al., 2008; Liu et al., 2008). In our study, protein contents varied significantly (p < 0.01) among the cell wall fractions and ranged from 8.44% to 27.6% (Table 1). The protein content of the supernatants S1, S2 and S3 were 33.9%, 23.2% and 22.4% on dry basis, respectively. The significant reduction of protein content from F2 to F3 is due to protease activity. A significant loss of protein was also observed during the lipase treatment, probably due to proteolytic coactivity, as noted by Jaehrig et al. (2008). Totally, about 95% of the proteins were released. This value is in agreement with results in the literature (Freimund et al., 2003). 3.4. Lipid content An elevated lipid content (12.6%) was observed in the yeast cell wall used. This would correspond to the presence of plasma membrane in the product (Dallies, François, & Paquet, 1998; Klis, 1994). This value was close to that reported by Freimund et al. (2003). Lipid contents varied substantially (p < 0.01) among F1, F2 and F3 but surprisingly there was no difference between F3 and F4 (Table 1). This means that the remaining lipids are not typical
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Table 3 Minerals contents (mg/kg dry weight basis) of fractions of the glucan isolation process. Fraction F1 F2 F3 F4 S1 S2 S3
P
K c
17804 ± 0.08 10448 ± 0.02b 5554 ± 0.00a 5295 ± 0.03a 1357 ± 24.2B 91.3 ± 4.19A 1468 ± 72.3B
Ca b
6035 ± 0.01 3144 ± 0.02ab 2768 ± 0.18ab 609 ± 0.00a 521 ± 3.76C 20.3 ± 0.08A 103 ± 2.02B
Na a
2030 ± 0.00 2954 ± 0.01c 2324 ± 0.00b 2299 ± 0.01b 8.78 ± 0.67A 11.7 ± 0.95A 8.17 ± 0.90A
Mg a
582 ± 0.00 9332 ± 0.02c 20274 ± 0.01d 7066 ± 0.01b 1474 ± 3.38C 217.06 ± 4.92A 1319 ± 4.84B
Zn c
837 ± 0.00 924 ± 0.00d 718 ± 0.00b 506 ± 0.00a 26.8 ± 0.11C 7.53 ± 0.07A 11.4 ± 0.09B
Cu a
271 ± 4.24 433 ± 8.49b 440 ± 1.41b 474 ± 1.41c 1.78 ± 0.05A 1.66 ± 0.06A 2.08 ± 0.03B
Fe a
5.62 ± 0.07 8.29 ± 0.28c 7.25 ± 0.07b 5.97 ± 0.07a 96.1 ± 18.5A 110 ± 0.33A 117 ± 1.77A
Mn a
47.78 ± 2.40 63.3 ± 4.10ab 64.1 ± 0.42ab 75.9 ± 8.06b 1.20 ± 0.18A 0.63 ± 0.04A 0.81 ± 0.01A
7.31 ± 0.21a 10.3 ± 0.28b 13.5 ± 0.35c 11.7 ± 0.78bc 0.11 ± 0.00A 0.18 ± 0.04A 0.23 ± 0.02A
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment.
Table 4 Total, a and b-glucan contents (% dry weight basis) of fractions of the glucan isolation process. Fraction
Total glucan
a-Glucan
b-Glucan
F1 F2 F3 F4 S1 S2 S3
15.6 ± 0.77a 32.2 ± 1.60b 41.5 ± 2.64c 54.4 ± 0.95d 11.12 ± 0.76B 20.5 ± 0.30C 4.98 ± 0.40A
2.17 ± 0.18b 2.21 ± 0.22b 1.03 ± 0.13a 0.99 ± 0.05a 5.25 ± 0.34C 3.08 ± 0.06B 0.62 ± 0.11A
13.4 ± 0.59a 30.0 ± 1.38b 40.5 ± 2.76c 53.4 ± 1.00d 5.90 ± 1.10A 17.5 ± 0.36B 4.36 ± 0.51A
Means ± S.D. Different small letters in the same column (a, b, c, d) indicate significant differences among sediment fractions (F1, F2, F3 and F4) at the p < 0.01 level, determined with Duncan’s test; different capital letters in the same column (A, B, C) indicate significant differences among supernatant fractions (S1, S2 and S3) at the p < 0.01 level, determined with Duncan’s test. F1 yeast cell wall (YCW), F2 YCW after hot water treatment, F3 YCW after hot water and protease treatment, F4 YCW after hot water, protease and lipase treatment, S1 supernatant after hot water treatment, S2 supernatant after hot water and protease treatment, S3 supernatant after hot water, protease and lipase treatment.
fats. In fact, it is known that, besides glycerides, several sterols and squalene are components of the lipid fraction in yeasts (Peña & Sandra, 1995). Blagovic´, Rupcˇic´, Mesaric´, Georgiú, and Maric´ (2001) found that squalene constituted 33% of total lipids in a brewer’s yeast. This might explain the apparent lack of lipase activity in our study. So, Magnani et al. (2009) reported that most of the b-D-glucan extraction processes do not include lipid extraction before enzyme treatment, since they may interfere with the protease action. Several organic solvents have been used for extraction of lipids during the removal of the b-D-glucan from S. cerevisiae. Liu et al. (2008) investigated extraction under reflux with isopropanol; however, Freimund et al. (2003) extracted only part of the lipids with the same solvent. Compared to our study, these extraction processes give low values of lipids (or traces).
3.5. Ash content and mineral composition Significant differences (p < 0.01) in ash contents were observed between fractions of the glucan isolation process. The ash content of b-glucan (2.94%) from baker’s yeast (S. cerevisiae) was higher than that of b-glucan from other sources, such as barley and oat cereals (1.42%, 1.56%; respectively) (Ahmad, Anjum, Zahoor, Nawaz, & Ahmed, 2010; Ahmad et al., 2009). Additionally, the significant amount of ash observed in supernatants might be due to process steps. This result is explained by increase in the solubility of minerals due to the number of washing proposed in this study.
Dietary fibre has long been known to form chelates with a number of divalent metals. These minerals have a capacity to modify functional properties of dietary fibre and offer a buffering action in solutions (Ahmad et al., 2010). The amounts of macro- and micro-elements detected in both supernatants and sediments of the cell wall fraction are shown in Table 3. The sediment of fraction F1 contains appreciable amounts of phosphorus, potassium and calcium. Moderate amounts of magnesium, sodium and zinc were also observed (837, 582 and 271 mg/kg dry weight basis). Minor amounts of iron, manganese and copper were also observed. In the present study, sodium, phosphorus and calcium were the dominating mineral components of extracted b-glucan (fraction F4). The amounts of zinc and iron in fraction F4 were 474 and 75.9 mg/kg dry weight basis. Contents of zinc and iron were approximately two times higher in the purified b-glucan powder than in the dry matter of the yeast cell wall extracts. Indeed, the abundance of iron (in higher concentrations in purified b-glucan sample) suggests that iron was associated with b-glucan rather than with phytate or proteins (Kivelä, Sontag-Strohm, Loponen, Tuomainen, & Nyström, 2011). The supernatant fractions S1 and S3 contained significant amount of minerals (Table 3). Phosphorus and sodium concentrations were the highest (1357–1468 mg/kg dry mass and 1319–1474 mg/kg dry mass, respectively) and modulate pH, followed in descending order by potassium (103– 521 mg/kg dry mass), copper (96.1–117 mg/kg dry mass), magnesium (11.4–26.8 mg/kg dry mass), calcium (8.17–8.78 mg/kg dry mass), zinc (1.78–2.08 mg/kg dry mass), iron (0.81–1.20 mg/kg dry mass) and manganese (0.11–0.23 mg/kg dry mass). Besides, the fraction S2 had only tiny amounts of P, K, Ca, and Na compared with other supernatant fractions (Table 3). Generally, 30% of sugars in S2 and S3 were not identified. This might be explained by the chosen conditions of acid hydrolysis. 3.6. Total, a- and b-glucan contents The content of total, a- and b-glucan of both sediment and supernatant of the cell wall fractions are shown in Table 4. The fraction with the lowest amount of glucan was the F1 fraction, which represents the native yeast cell wall. The highest glucan content (54.4%) was in the fraction F4 when hot water, protease and lipase treatments were applied for extraction of b-glucan. The significant increase in glucan content from fractions F1 to F4 is due to the extraction and the purification steps of b-glucan. Thus, our process steps gave better yield and higher purity than the traditional isolation process involving treatments with hot alkali and acids. The supernatant fraction with the highest amount of glucan was the S2 fraction. Statistical analyses show significant differences (p < 0.01) in total glucan content among both sediments and supernatants of the cell wall fractions. Actually, these differ-
C. Borchani et al. / Food Chemistry 163 (2014) 108–113
ences could be explained by the way the enzyme works. In the other words, the extraction process used in our study showed a significant difference in total glucan content due to the extraction steps comprising the hot water and the Savinase and Lipolase enzyme activities. 4. Conclusions In the present paper, an enzymatic isolation process adapted from Jaehrig et al. (2008), has been established for producing valuable b-glucan from baker’s yeast. Our process has some distinct advantages compared to previously published isolation processes using the solvent treatment. First, the soft treatments with water and enzymes avoid chain degradation and therefore preserve the original glucan structure of the cell wall. Second, b-glucan was obtained in a yield of 18.0% (w/w) of the original ratio in the yeast cell walls and a purity of 79.0%. Finally, the choice of yeast isolation process appeared to be important as it affected most of the chemical properties e.g. mineral and total glucan contents among the sediment and the supernatant fractions. Enzymatic treatment steps with Savinase and Lipolase enzymes were found to be good since they gave high b-glucan content, but also removed more ash, fat and protein during the extraction of b-glucan. In conclusion, the isolation process used in this study is inexpensive and it is easy to produce b-glucan from S. cerevisiae for industrial utilisation. References AACC (2003). Approved methods of American Association of Cereal Chemists. St Paul: The American Association of Cereal Chemists Inc.. Ahmad, A., Anjum, F. M., Zahoor, T., Nawaz, H., & Ahmed, Z. (2010). Extraction and characterization of b-D-glucan from oat for industrial utilization. International Journal of Biological Macromolecules, 46, 304–309. Ahmad, A., Anjum, F. M., Zahoor, T., Nawaz, H., & Din, A. (2009). Physicochemical and functional properties of barley b-glucan as affected by different extraction procedures. International Journal of Food Science & Technology, 44, 181–187. AOAC (1990). Official methods of analysis (15th ed.). Arlington: The Association of the Official Analytical Chemists. Blagovic´, B., Rupcˇic´, J., Mesaric´, M., Georgiú, K., & Maric´, V. (2001). Lipid composition of brewer’s yeast. Food Technology and Biotechnology, 39, 175–181. Blakney, A. B., Harris, P. J., Henry, R. J., & Stone, B. A. (1983). A simple and rapid preparation of aldithol acetates for monosaccharide analysis. Carbohydrate Research, 113, 291–299. Bohn, J. A., & BeMiller, J. N. (1995). (1 ? 3)-b-D-Glucans as biological response modifiers: A review of structure-functional activity relationships. Carbohydrate Polymers, 28, 3–14. Cabib, E., Dong-Hyun, R., Schmidt, M., Crotti, L. B., & Varma, A. (2001). The yeast cell wall and septum as paradigms of cell growth and morphogenesis. Journal of Biological Chemistry, 276, 19679–19682. Dallies, N., François, J., & Paquet, V. (1998). A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast, 14, 1297–1306.
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