Canadian Journal of Microbiology
Issued by THE NATIONAL RESEARCH COUNCIL OF CANADA
VOLUME 1 DECEMBER 1954 NUMBER 3
YEASTS OCCURRING ON APPLES AND IN APPLE CIDER1
BY D. S. CLARK,". H. WAI,LACE,~ AND J. J. DAVID"
Abstract
A s t u d y w a s ~ n a d e o f t h e predominant yeasts t h a t occur o n apples grown i n Quebec, and i n cider. T h e yeasts isolated fr0111 apples were members o f t h e family~Cryptococcaceae and o f t h e genera Candida, Cryptococczu, RIzodotorrrla, and Torulopsis. Species o f Candida were b y far t h e m o s t predo~ninant . T h e yeasts isolated f r o m cider were representatives o f t h e genera Debaryonzyces, Pichia, and Saccl~aro~nyces. All t h e yeasts studied were classified i n t o species o f t h e respective genera.
In recent years many of the predominant yeasts occurring on the surfaces of various fruits have been isolated and classified (2, 6, 16, 17, 18, 21). I t seems, however, that very few reports have been published about yeasts indigenous to apples; Lewis (12) isolated Endonzyces ma l i from decaying apples; Grosbusch ( lo) , Torz~la rubesfaciens from apple parings; and Lodder and Kreger-van Rij (13), Candida krusei from an apple. Information concerning the predominant yeasts occurring in apple juice and cider seems also to be scarce; Osterwalder (19) isolated Saccharo?nyces heterogenicus and S. micro- ellipsodes from apple juice; Lodder and Icreger-van Rij (13), S. elegans; and Marshall and Walltley (15), several species of Rhodotorula, Saccharomyces, and Torz~lopsis. Guilliermond and Tanner (11) described two strains of S. ma l i isolated from apple cider; Pearce and Barker (20) studied the yeast flora of sweet and black cider and described 13 species of yeasts.
I t seems that a taxonomic study of the yeasts occurring on apples and in apple products is timely; the worli reported in this paper was done, therefore, to determine what yeasts predominantly occur 011 apples grown in the province of Quebec, and in cider.
iMarzz~script YeceiVcd Arrgzrst 9 , 193'4. C07itr~bi~tio~z froin ~ J L C Faczrlty of Agricidtzrre, illcGil1 University, Macdonald College,
Qzrebec, Canada; J[acdonald College Jozrmal Series No. 357. Lectzrrer, Departnzent of .4griczrltr~ml Bacteriology.
3 Associate Profcssor, Departl;zc?~t of Agriczrltzrral Bacteriology. " lss is ta~zt Profcssor of Ilortictrltrrrc, and Food Tecl~nologist, Qzrebcc Departnzent of
d gricultzrre.
[ T h e October number o f th i s Journal ( C a n . J . Microbiol. 1 : 85-143. 1954) w a s issued October 8, 1954.1
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146 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 1
Experimental Methods
Isolations from Afifiles Twenty-two samples of apples were collected in October, 1952, a t two
widely separated places in Quebec. At Macdonald College six samples were gathered randomly from crates containing freshly picked McIntosh apples, and a t Franklin Center 16 samples of wild apples were collected from several trees. In each instance sterile forceps were used to carry the apples by their stems to large sterile culture dishes. In the laboratory each apple was quartered rapidly by means of a sharp knife, placed with 400 ml. of water in a Waring Blendor bowl, and comminuted four times for 30 sec. a t one- minute intervals. The suspensions were diluted to 1 : 10, 1 : 100, 1 : 1000, and 1 : 10,000 with sterile distilled water; five 1-ml. replicates of all the dilutions were plated with apple juice agar (14). The plates were incubated a t 25" C. for four days and then examined for yeasts that appeared to be different from one another. Fifty of the most predominant yeasts were isolated, purified by serial plating, and maintained on slopes of a yeast cultivation medium described by Wickerham (22).
Isolations from Cider Two 400-ml. samples were taken from the taps of each of two large barrels
of apple cider. The juice for this cider had been pressed from a mixture of wild apples, treated with sulphite, and transferred to large barrels where i t was allowed to ferment and mature under natural conditions for one year. Ten milliliters of each of the four samples were pipetted into flasks containing 100 ml. of sterile apple juice. After the cultures had incubated for four days 10 ml. of each were used to make 1 : 1000, 1 : 10,000, 1 : 100,000, and 1 : 1,000,000 dilutions with sterile distilled water. One milliliter of all dilutions was plated on apple juice agar and incubated for four days. Ten yeasts that appeared morphologically different from one another were isolated, purified, and maintained on slopes of yeast cultivation medium.
Taxonomy With a few exceptions, the procedures followed and media used for taxonomic
studies were those described by Lodder and Kreger-van Rij (13) and Wickerham (22). A modification of a method described by Wickerham and Duprat (23) was used for the examination of pseudo and true mycelia, blastospores, and arthrospores. Petri plates each containing two glass slides were sterilized by dry heat; Wickerham's yeast morphology agar (22) was poured into one set of the plates until it covered the slides to a depth of 1 mm. ; Benham's corn meal agar was used for another set of the plates. When the agar had solidified, cells of the yeasts were streaked in a straight line on the agar over the slides; sterile coverslips were placed over a portion of the streaks and the cultures were incubated for five days a t 25' C. The slides were
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CLARK ET AL.: YEASTS ON APPLES 147
removed from the Petri plates for observation, the agar removed from beneath them, and the growth occurring under and beside the coverslips studied under a microscope.
Seven different media were used to test for the development of ascospores, namely: carrot slices, Gorodokowa's agar, gypsum blocks, malt extract agar, one-sixth strength tryptone glucose agar, and vegetable juice agar. The tryptone glucose agar was prepared by dissolving 6 gm. of Difco tryptone glucose agar and 11.5 gm. of agar in 1000 ml. of distilled water (9). The sodium acetate agar was prepared as described by Adams (1). A method recommended by Gray (8) was used for staining ascospores. The liquid- culture and auxanographic methods were both used for the carbon and nitrogen assimilation tests.
Results and Discussion
Sixty isolated yeasts were studied. Approximately 75% of those occurring on wild and cultivated apples appeared very much the same; 27 of these were isolated and found to have identical microinorphological and physiological characteristics. These characteristics were unlike those of any previously described species; consequently the yeast was given the binomial Candida malicola (4). a
The names of the species studied, their origin, and their occurrence are given in Table I. I t is interesting to note that all the yeasts isolated from apples were members of the family Cryptococcaceae. In addition to their
TABLE I
SPECIES or; YEASTS ISOLATED FROM CULTIVATED AND WILD APPLES, AND FROM APPLE CIDER
Source Yeasts No. of cultures isolated
Cultivated apples Cntrdidn ~ i~nl ico ln Torrllopsis fn f i~a la Rl~odolorr~ln glz~liizis var. rz~bescens Rhodolorr~ln iirz~ciliginosa Cryplococcr~s nlbidus Cryplococcus rreoforinans
Wild apples Candida ?nnlicoln Cryplococcus albidr~s Cryplococcz~s lar~re t~ t i i Cryplococcz~s neofornzans Candida scollii
Apple cider Pichia nze~i2branaefaciens 2 Sacckaro?~zyces ovifororn~is 2 Saccharoi~zyces cerevisiae 1 Saccharoi~zyces steineri 1 Debaryot~zyces kloeckeri 1 Torz~lopsis candida 1 Ca~zdida r~zesenterica 1 Pichia polynzorpha 1
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148 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 1
inability to form ascospores these yeasts were not able to ferment any of the common sugars. They were classified into seven genera and 16 species by means of the schemes of Lodder and Icreger-van Rij (13). The asporogenous yeasts were represented largely by Candida malicola and to a lesser extent by species of Cryptococcus, Rhodotorula, and Torulopsis. The perfect yeasts were represented by species of the genera Debaryofnyces, Saccharomyces, and Pichia. Candida malicola, Cryptococcus albidus, and Cryptococcus neoformans were common to both cultivated and wild apples. The perfect yeasts found in cider produced abundant and typical ascospores but only the Saccharomyces species were capable of fermenting glucose.
Since 27 of the 50 yeasts isolated from apples were identified as Candida malicola, i t is very probable that this yeast is indigenous to Quebec apples. This yeast was found again in great numbers on McIntosh apples in 1953.
I t is probable that the results of this work do not show all the types of yeasts tha t are indigenous to apples grown in Quebec. I t may be that they represent a group of yeasts tha t are not easily removed from the surface of apples by weather. I t was possible to acquire information about the weekly amount of rainfall from only one area, Macdonald College, where a total of 2.84 in. of rain fell during the first week of October, 1952. Since the apples were gathered shortly after this time, i t is reasonable to believe that a great many types of microorganisms could have been washed off by the rain. Marshall and Walltley (14) have shown that rainfall considerably reduces the number of yeasts on apples. They reported that the average numbers of yeasts for the months of September and October were 2934 and 424 per c m 2 , respectively; the rainfalls were 1.96 and 6.62 in., respectively. Charley (3) stated that appreciable infection of apples by wild yeasts occurs in early autumn and tha t a large proportion of those additions to the microflora of apples can be removed by washing. Furthermore, Forgacs (7) showed tha t rinsing apples in tap water reduced the number of mesophilic organisms from 252 to 13 per 4 sq. in. of epidermal surface.
Tlle fact that three of the species identified in the present work were members of the genus Cryptococcus strengthens the belief that the yeasts isolated were in indigenous group. Cells of species of Cvyptococcz~s are surrounded by a starch-containing capsule, a characteristic that causes them to clump together and give a positive starch-iodine test (22). I t is assumed tha t such capsules would probably aid yeasts in remaining attached to the surface of apples, regardless of the washing effect of heavy rains.
Certain well-defined species of yeasts have been isolated from bottled cider (20), but no evidence exists to show tha t those yeasts may be especially asso- ciated with certain apples. I t is interesting to note, however, that over 50% of the cultures isolated from the cider used in this work were non- fermenters and tha t all the yeasts isolated from the apples were also non- fermenters. I t has been noted elsewhere (5) that the presence of the non- fermenting yeasts in apple juice apparently does not influence the fermenting activity of the Saccharomyces species.
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CLARK ET AL.: YEASTS ON APPLES 149
Acknowledgment
The authors wish to thank the Scientific Research Bureau, Quebec Depart- ment of Trade and Commerce, for a grant to defray the expenses of this investigation; they are also indebted to Mr. Fernand Dufour, Franklin Center, Quebec, for supplying apples and cider.
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