Fermentation of wheat grain by Schwanniomyces castellii

Biomass 15 (1988) 175-185

Fermentation of Wheat Grain by

Schwanniomyces

castellii R. Z a i r e , G. M o u l i n , * E G a l z y Chaire de G6n&ique et de Microbioiogie, ENSA, 34060 Montpellier C6dex, France

J. Herard, G. Deshayes & B. Godon Laboratoire de Biochimie et de Technologie des Prot6ines, INRA, rue de la G6raudi6re, 44072 Nantes CEdex 3, France (Received 8 October 1987; revised version received 4 January 1988; accepted 12 January 1988)

ABSTRACT Fermentation of flour and wheat grain, without enzymatic hydrolysis has been demonstrated using a strain of Schwanniomyces castellii. Ethanol production yields were fairly good although starch was not totally degraded. The composition of the fermentation residues was compared to that of distillers dried grains. Key words: Schwanniomyces castellii, wheat, fermentation, yeast, alcohol. INTRODUCTION Direct alcoholic fermentation of flour and of crushed wheat requires the use of a strain of yeast which possesses an amylase activity and which is capable of fermenting the products of hydrolysis. Schwanniomyces castellii CBS 2863 was selected from among the strains tested by Frelot et al. ~ Previous studies have shown that it possesses a-amylase and amyloglucosidase activities 2 and that oxygen plays a decisive role in the biosynthesis of amylases. 3 A respiratory-deficient mutant (DR 12) has 175 * To whom all correspondence should be addressed.

Biomass 0144-4565/88/S03.50 - © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain

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R. Za~'re, G. Moulin, P. Galzy, J. Herard, G. Deshayes, B. Godon

been obtained from this strain, which ferments in the presence of air.4 Trials carried out on wheat flour showed that prior gelatinization was necessary. In the present work, the fermentation of two products of wheat with different compositions and grain sizes has been studied and comparative analysis carried out on raw material and fermentation residues.

MATERIAL AND METHODS

Biological material Yeast strain

A respiratory-deficient mutant (DR 12) of the strain Schwanniomyces castellii was used. This strain has lost cytochrome-b; however, it has retained the alternative respiratory pathways. 5

Culture conditions Preliminary culture was carried out at 30°C using an oscillating mechanical agitator (80 osc. per min, amplitude 8 cm) in an Erlenmeyer flask filled to a tenth of its volume and containing soluble starch (50 g/litre) and yeast extract (10 g/litre). This medium was buffered to pH 5.4 (tartrate M/20 disodium phosphate M/10 buffer). The fermentation medium consisted of 75 g of the wheat starch material, 3 g of NH4H2PO 4 and 1 g of (NH4)2SO 4 in 50 ml of distilled water and 350 ml of pH 5.4 buffer. It was autoclaved at 105°C for 20 min and then agitated strongly to break up the gel, aerated and cooled to 32°C. The medium was inoculated at this temperature, with 100 ml of the culture (i.e. 500 mg dry matter of DR 12 yeast and 200 units of aamylase activity).

Raw materials Two fermentation substrates were used: Firstly a mixture (M) of milled material (flour, middlings and small bran) prepared in a Brabender Senior laboratory mill (Brabender, Hackensack, New Jersey) and composing 85% of the grain. Secondly crushed material (B) produced using a SOCAM mill without a screen but set to obtain a particle size of 1150/~m.

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Analytical techniques Estimation of cell population The number of cells was determined by counting on a THOMA cell. This method gave 106 cells in 1 mg of dry matter. Sugar assays Starch and soluble sugars were assayed together using the method described by Bergmeyer et al. 6 and after the successive actions of amyloglucosidase and glucose-oxidase according to the method of Thivend et al. 7 It should be noted that autoclaving the raw material at a concentration of 10 g/litre at 120°C for 20 min is indispensable before hydrolysis. However, autoclaving is not necessary for the inoculum and for the fermenting medium; heating to 100°C for 15 rain is sufficient to kill the yeasts. Ethanol assay Ethanol was assayed by gas phase chromatography. Evaporated ethanol was first recovered by bubbling fermentation gases through a series of Erlenmeyer flasks containing sterile water at 3°C. Amylase assay Amylase activity was assayed using the method previously described. 2 Polysaccharide assay Parietal polysaccharides in milled substances were assayed after enzymic amylolysis followed by proteolysis in the case of fermentation residues using the method described by Brillouet et aU Protein assay Total nitrogen was determined by the Kjeldahl method. The results are expressed as the percentage of proteins after multiplication of the preceding values by a coefficient of 5.7. Ash assay The amount of ash was obtained by mineralization of the substance at 900°C. Lipid assay Determination of the total fats content was carried out in accordance with ISO standard 7302.

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A m i n o acid assay This determination was carried out using a Kontron apparatus after acid hydrolysis of proteins. The results are expressed as the number of residues of an individual amino acid per 1000 residues of total amino acids.

Protein solubility Solubifity in water 30 ml of distilled water and 1 g of freeze-dried draft were placed in a glass tube with a ground glass stopper. The tube was fixed to a rotary agitator (1 rpm) at ambient temperature for 15 min. The contents of the tube were then centrifuged at 6000g for 20 min at a temperature of 18 to 20°C. The soluble nitrogen present in the extract was assayed after settling. The operation was carried out in duplicate. Solubility in N a O H N/20 15 ml of NaOH N/20 solution was added to the tube containing the pellet from the centrifugation above. It was agitated for 1 h on the same rotary agitator and centrifuged as above. A second extraction operation with 15 ml of NaOH N/20 was carried out with agitation for 30 min and centrifugation. Nitrogen from the two extracts and the extraction residue was assayed in order to draw up a full solubilisation balance.

High performance liquid chromatography This was carried out firstly with gel filtration and also in reverse phase using KONTRON 600 apparatus (KONTRON HPLC system monitored by a 200 series programmer).

Gel filtration Elution was carried out in a column (10 m m x 280 ram) filled with Superose 12 attached to beaded agarose. The eluting agent was an aqueous solution of acetonitrile (80/20 v/v) to which was added 0.1% of trifluoracetic acid (TFA).

Reverse phase chromatography This type of chromatography was carried out using a column (4"6 mm x 250 mm) of Ultropac TSK ODS 120T 5 mn. An elution gradient

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179

was obtained by mixing a solution (A) (U20 + 0"1% TFA) and a solution (B) (H20 + acetonitrile 20/80 v/v + 0-1% TFA) as follows: 6 min at 0% (B); 70 min from 0 to 100% (B); 5 min at 100% (B); 10 min from 100 to 0% (B). The volume of the deposit was 100 nl.

RESULTS AND DISCUSSION Alcoholic fermentation

Fermentation was carried out in a 2-1itre fermenter for 52 h at 30°C. Aeration and agitation were fixed at 1 vvm (volume air/volume medium/ minute) and 240 rpm respectively for the first 24 h. They were then kept at 0.3 vvm and 120 rpm until the end of fermentation. Fermentation was stopped after 52 h since beyond this period the residual substrate was no longer attacked because of the decrease in amylase activity. This decrease during fermentation has also been reported elsewhere. 9 The fermented material was centrifuged (10000 rpm for 10 min). The ethanol, residual sugars and soluble dry matter contents were determined in the supernatant. The centrifugation pellet (fermentation residues) containing yeasts and residual insoluble constituents of wheat was analysed (Table 1). The data show that the carbohydrate fraction was not completely utilised since amylase activities, and particularly a-amylase activities, decreased during fermentation. The centrifugation supernatant contained approximately 30% of residual total dry matter. This supernatant consisted of mineral salts (14.4 g/litre), carbohydrate (3.5 to 4 g/litre) and soluble wheat proteins (4.3 to 6"6 g/litre), which could be recovered. Production of ethanol represented 67% and 52% of the theoretical conversion yield of consumable sugars for substances M and B respectively. It should be noted that this is the apparent yield; the sugar used contributed partly to the formation of ethanol and partly to growth of the yeasts (approximately 5 g/litre of dry matter). A further fermentation trial was carried out under the same conditions using fibre-less wheat flour (data not shown). Considerable retrogradation was observed after autoclaving; only 50% of the starch was fermented. The conversion yield obtained was approximately 52% of the theoretical conversion yield of the sugars used up. It would therefore seem that the presence of fibres reduces the consequences of retrogradation and facilitates the attack of starch.

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R. Zai're, G. Moulin, P. Galzy, J. Herard, G. Deshayes, B. Godon

TABLE 1 Composition (g/litre) of the Fermentation Substances Products

Dry matter Initial sugars Yeast dry matter Dry matter of the fermentation residues Dry matter of the supernatant Residual sugars Ethanol

Material Ma

Bb

164 101" 1 4.6 52.5 29.5 17.1 28"4

164 97.4 4.7 59.3 26"3 18.4 21.3

aMixture of milled material. bCrushed material.

Analysis of fermentation residues of substances M and B

The fermentation residues obtained (Table 2) still contained fermentable carbohydrates and displayed the features of DDG (Distillers Dried Grains) of other origins. Fermentation residues of substances M and B had relatively low soluble protein contents (Table 3). It was shown that these proteins were present in the centrifugation supernatant under the experimental conditions used. The traditional method is to distill the fermented substances and then to dry the residual liquor to obtain DDG. Under these conditions it is normal to find soluble proteins in the dried substance. Although the laboratory conditions used do not allow direct comparison of the fermentation residues with DDG, they allow comparison of the composition of each soluble and insoluble part. Reverse phase chromatography using a TSK ODS 120T column with an acetonitrile gradient indicates that fermentation residues of substances M and B contain proteins which are more hydrophobic than those of DDG. Apart from the first three peaks which resemble those of the latter, the other peptide groups were eluted by a 30 to 50% concentration of acetonitrile instead of 15 to 35% (Fig. 1). In addition, the results of gel filtration show that the two fermentation residues studied contained groups with a higher apparent molecular weight than usual: 62 000 D instead of an average of 54 000 D (Fig. 2). It should be noted that a fraction of the proteins was not recovered (soluble proteins) under the experimental conditions used.

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TABLE 2 Chemical Composition of Mixture of Milled Material (M) and Crushed Material (B) and Corresponding Fermentation Residues (% wt/wt Dry Matter) Product

Proteins (N × 5.7) Total lipids Starch and soluble sugars Parietal polysaccharides Ash

Material M

Fermentation residues o( M

B

Fermentation residues of B

12.0 2.0 76-6 8'2 1.1

29.1) 5"0 32.9 27.5 5"5

12"0 2'0 73"8 10.4 1.7

29.7 5.4 28.1 30"3 6.5

TABLE 3 Solubility of Nitrogenous Compounds of Fermentation Residues in Comparison with DDG of Wheat and Grain of Wheat Grain of wheat

Total nitrogen (% Dry weight) Solubility of proteins (% N total) In water In NaOH Insoluble Indefinite

DDG of wheat

Fermentation residues

M

B

2.1

5'9

5-1

5-2

20.0 55.0 25-0 0

20.7 19"8 46-0 13'5

5-6 28.8 58"7 7.1

5.5 32.1 51.0 11.4

The amino acid compositions of M and B fermentation residues were compared with various substances (Table 4). It is notable that there are considerable differences between the substances obtained. The fermentation residues had a higher cysteine content than that of whole egg.l~ The other essential amino acids are limiting, but are generally present in larger quantities in D D G and in substance M than in wheat. Like most yeasts, Schwanniomyces castellii is rich in lysine; the presence of a large quantity of yeast cells in fermentation residues causes an increase in the lysine content both in percentage of amino acids present and in percentage of total dry matter.

R. Zai're, G. Moulin, P. Galzy, J. Herard, G. Deshayes, B. Godon

182

Grod=ent %B

O. D.

100

80

./

60

O,S 40

20

Volume

Gradlenb

O.D.

%a 100

80

60 O,S

Sample B 40

20

Volume

Fig. 1. Reverse phase chromatography column of ultrapac TSK a D S 120T. Elution gradient indicated in percentage of solution (B) (acetonitrile 20/80 v/v + 0.1% trifluoroacetic acid).

Fermentationof wheatgrain by Schwanniomycescastellii

183

O.D.

o,s Sample M

I

6 7 000

45 000 Molecular ,weight

'

@.D.

0,s

I

I

67 000

4 S000 Molecular weighl

Fig. 2.

Gel filtration in a column filled with Superose 12. Elution by an aqueous solution of acetonitrile(80/20 v/v) and 0"1%of trifluoraceticacid.

The respiratory-deficient mutant (DR 12) of Schwanniomyces castellii hydrolyses and ferments flours and autoclaved crushed wheat grain. The presence of fibres reduces the effects of the retrogradation phenomenon. Ethanol production yields are fairly good but starch is not totally degraded. This results in fermentation residues which are richer in starch than traditional substances, which can be advantageous for certain utilisations. The proteins of these fermentation residues, which are enriched in lysine because of the presence of yeasts, have slightly higher molecular weights than the proteins of D D G and possess more hydrophobic amino acids.

184

R. Za't're, G. Moulin, P. Galzy, J. Herard, G. Deshayes, B. Godon TABLE 4

Amino Acid Composition (Residues per 1000 Residues) of Fermentation Residues in Comparison with Different Products. Amino acids

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Cysteine Methionine

Fermentation residues M

B

69.7 32.5 38.8 379.6 82"9 55.6 39.4 25 23.2 34.8 36.2 32"5 18.3 25"4 111.3 37.7 24.8

57"7 50.8 40 154"6 115"2 38'3 28.5 32.8 54"7 111.7 32.3 61.7 35"9 81.2 79'1 85"9 28'3

DDG of wheat

Wheat

S. castellii ~

Egg b

66 34.9 51.5 364.8 108"3 48"1 41.4 40.8 30'6 79.2 35"3 53"7 31 26.9 52.9 33.0 19.9

32 25 53 325 133 56 36 56 40 71 18 41 13 17 28 30 20

88.7 42 46.3 181.9 43 48.1 83.6 56.9 47.5 70 14.9 55.1 93"7 17"1 53"4 18-2 9-9

-51 -----73 66 88 42 58 64 --24 31

~From Ref. 10. bFrom Ref. 11.

ACKNOWLEDGEMENTS W e t h a n k M. X. R o u a u o f the l a b o r a t o i r e d e B i o c h i m i e et t e c h n o l o g i e des glucides f o r p o l y s a c c h a r i d e s analysis. T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m A F M E ( A g e n c e f r a n c a i s e p o u r la m a i t r i s e d e l'6nergie).

REFERENCES 1. Frelot, D., Moulin, G. & Galzy, P., Strain selection for the purpose of alcohol production from starch substrates. Biotechnol. Lett., 4 (1982) 705-8. 2. Oteng-Gyang, K., Moulin, G. & Galzy, E, A study of the amylolytic system of Schwanniomyces castellii. Z. All. Mikrobiol., 21 ( 1981) 5 3 9 - 4 6 .

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3. Boze, H., Moulin, G. & Galzy, E, Influence of environment on the cell yield and amylase biosynthesis in continuous culture by Schwanniomyces castellii. Archives of Microbiology, 148 (1987) 162-6. 4. Poinsot, C., Moulin, G., Claisse, M. & Galzy, P., Isolation and characterization of a mutant of Schwanniomyces castellii with altered respiration. Antonie van Leeuwenhoek, 53 (1987) 65-75. 5. Poinsot, C., Boze, H., Moulin, G. & Galzy, E, Respiratory pathways in Schwanniomyces castellii. Biology of the cell, 58 (1986)65-70. 6. Bergmeyer, H. U., Bernt, E., Schmidt, E & Stork, H., D-glucose determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Methods of enzymatic analysis, Bergmeyer, H. U. (ed.), Vol. 3, Academic Press, London, 1974, pp. 1196-201. 7. Thivend, E, Mercier, C. & Guillot, A. (1965). Dosage de ramidon dans les milieux complexes. An. Biol. Anita. 5 (4), pp. 513-20. 8. Brillouet, J. M., Rouau, X., Hoebler, C., Barry, J. L., Carre, A. & Lorta, E. A new reference method for determination of insoluble cell walls and soluble non-starchy polysaccharides from plant materials. J. of Agric and Food Chem. (in press). 9. Malfait, M.-H., Moulin, G. & Galzy, E, Ethanol inhibition of growth fermentation and starch hydrolysis in Schwanniomyces castellii. J. Ferment. Technol., 64 (1986) 279-84. 10. Rossi, J. & Clementi, E Protein production by Schwanniomyces castellii on starchy substrates in liquid and solid cultivation. J. of Food Technology, 20 (1985)319-30. 11. FAO/WHO Expert Group on Protein Requirements, FAO Nutrition Meeting Report Series No. 37; WHO Report Series No. 230, 1965