March, 1935
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349
duction was due chiefly to lower prices; the quantity h- refined natural, $670,000 and $479,000; synthetic, 3258,000 ported, 2,361,000 pounds, was only about 40 per cent under and $220,000. Exports of naval stores, gums, and resins were slightly that of the year before. under those of 1933 in value. The trade of the last two years Gum, RE~INS AND , Ka1-a~STORES is shown in Table VII. A large increase in imports of shellac made it the leading OF XAYAL STORES, GT'MS, AND RESINS article in the gums and resins group in point of value, surpass- TABLEVII. EXPORTS (IX THOUSANDS) ing chicle, although the chewing gum material also registered -19331934--a good gain. Unbleached shellac was imported to the exQuantity Value Quantity Value tent of $1,686,000 in 1934; bleached shellac to a value of Naval stores: 994 8 6,540 794 8 6,615 Gum rosin, barrels $126,000; and seed lac, crude lac, etc., to a value of $1,127,231 219 1,324 1,779 Wood rosin, barrels 9,789 13,388 5,781 4,755 G u m spirits of, turpentine, gallons 000. These totals compared with $906,000, $66,000, and 893 403 343 851 Wood turpentine, gallons 9 65 98 7 T a r and pitch of wood, barrels $873,000 in 1933. Chicle imports were 6,499,000 pounds as 661 6.000 839 4,248 gums and resins, pounds compared with 4,066,000 and their value $1,471,000 as com- Other 14,714 1 4 , 4 89 Total pared with $1,081,000. Camphor imports in 1934 and 1933 were, respectively: crude natural, $646,000 and 8411,000; RECEIVED February 4. 1935.
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Aerobacter aerogenes as a Cause of Ropiness in Maple Sirup F.W. FABIAN AND H. H. B~JSKIRK, Michigan State College, East Lansing, Mich.
R
OPY fermentation is not
uncommon i n c e r t a i n products such as milk, bread, and wines. It is sometimes found in such factories as those making sugar and molasses. This type of fermentation may occur a t any season of the year but is more common in the spring and fall. Stark and Foster ( 3 ) have shown that a large number of different bacteria are capable of producing this type of fermentation. They are omnipresent in the soil, in the air, on feeds, and in the dust which collects in the barn, dairy, or factory. Therefore, the likelihood of ropy fermentation occurring in certain of the food industries is great.
ROPYMAPLESIRUP
producing a ropy fermentation in normal concentrated sirup. Kormal maple sirup was then diluted to various concentrations. M e m b e r s of t h e s i x groups of bacteria previously isolated were inoculated into the various dilutions, and they were found to grow well in a concentration of maple sugar u p to 10 per cent. Above that point the g r o w t h was slow or absent. After 24 to 48 hours of incubation a t room temperature, the dilute sirup was again concentrated. Of all the b a c t e r i a tested, only those in group I produced ropiness. The bacteria in this group within 24 to 48 hours, depending upon the amount of i n o c u l u m u s e d , were able to change the sugars p r e s e n t i n the dilute maple sirup or sap so that it was e x t r e m e l y r o p y when evaporated to the concentration of normal maple sirup. The viscosity was so great t h a t it was possible to take a loop, dip i t into the concentrated sirup, and stretch it 10 feet or more before the thread would break.
A group of bacteria was isolated f r o m the sap of iicer saccharum which, when inoculated into sterile sap or dilute maple sirup, produced a ropy maple sirup upon being concentrafed to the consistency of sirup. Since these bacteria were isolated f r o m the sap f r o m which the ropy maple sirup was produced in the sugar bush, it is evident that t h ( y were responsible f o r the condition. The morphological, physiological, and cultural characteristics of the bacteria responsible f o r this condition corresponded in all essential details to those of Aerobacter aerogenes. The addition of a n amount of acetic acid of approximately the acidity f o u n d in the fermented sap did not influence the consistency of the evaporated sap. T h e addition of a similar amount of luetic acid did inJuence its consistency. Neutralizing thc acidity of fermented sap reduced somewhat the ropy condition of the concentrafed sap.
I n the spring of 1934 sei-era1 m a k e r s of maple sirup were troubled with a ropy product. Samples were brought to the laboratory for analysis. T h e concentrated maple sirup was examined for the presence of microorganisms, both microscopically and by plating i t on solid media-yeast extract, malt extract, dextrose, and plain agar. These media were seeded with large and small amounts of maple sirup, but in no instance did growth appear. Samples of sap were then procured from the sugar bushes from which the ropy inaple sirup was being produced. Samples of this sap in different stages of concentration were examined and plated on the media just mentioned. All such samples showed the presence of abundant bacteria. Many representative colonies were fished on agar slants and purified by repeated platings. These colonies were then classified into six groups (Table I). S o n e of the organismb isolated was capable of growing or
BACTERIS
RESPOXSIBLE FOR ROPIKESS
After repeatedly testing the various groups for their ability to produce r0pine.s in various concentrations of maple sirup, i t was found that group &I.S. 1 was responsible. The members of this group were then studied morphologically, culturally, and physiologically in a n attempt to identify them. A complete description of the organisms follows: Morphological characteristics: Size and shape: rods, 0.5 to 0.8 by 1.5to 2.5 microns, occurring singly, in pairs and sometimes short chains, asporogenic, encapsulated, motile in broth and sap, gram-negative
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INDUSTRIAL AND ENG NEEHING CHEMISTRY
Cultural characteristics: Gelatin colonies: smooth, opaque, moist, white, entire Gelatin stab: no liquefaction; white, thick, spreading, opaque surface growth Agar colonies: surface colonies thick, white, raised, moist, smooth, entire, subsurface lenticel Agar slant: abundant, thick, white, moist, glistening, mucoid growth Broth: turbid, with ring formation Physiological characteristics: Litmus milk: acid coagulation with gas formation, slight reduction, no peptonization Potato: yellowish white to yellowish brown, spreading, with nodular outgrowths over the surface Indol not formed Nitrates reduced to nitrites; some ammonia produced Acetyl methylcarbinol formed Methyl red negative Uric acid positive Sodium citrate positive Acid and gas in glucose, fructose, galactose, lactose, arabinose, xylose, mannose, maltose, sucrose, rhamnose, raffinose, mannitol, sorbitol, dulcitol, salicin, glycerol, and trehalose; no fermentation in inulin, dextrin, adonitol, and inositol Ratio of COSto Ht is 2 to 1 Aerobe, facultative Optimum temperature, 25’ C. Habitat, isolated from maple sap Checking the characteristics of these bacteria with the various genera of bacteria found in Bergey’s manual (I), it was found that they corresponded closely with those of Aerobacter aerogenes. There are some points of difference, however, which should be noted. The most striking is that of motility. The bacteria in group I were actively motile a t the end of 24 hours in plain broth and in broth to which maple sirup had been added. In this latter broth they gradually lost their motility because of the formation of capsules. When grown on dextrose agar, they were nonmotile after a short time for the same reason. The second point of difference was in the formation of a capsule. The members of group I were encapsulated. This could be readily demonstrated by the capsule stain.
Vol. 27, No. 3
the characteristics of the present writers’ organism reveals fundamental differences which would not warrant classifying it as B. aceris.
INFLUENCE OF ESCHERICHIA-AEROGESES GROUPON MAPLESIRUP
To study the influence of the members of the Escherichia-
derogenes group on sap and dilute maple sirup, several laboratory cultures were inoculated into these media. One culture of Escherichia coli and four cultures of Aerobacter aerogenes from various sources were used. The cultures were checked and found to be true to type. The results of this experiment are recorded in Table 11. Two cultures from group I were inoculated into the same material as a control. TABLE11. EFFECTOF GROWING DIFFERENTORGANISMS IPU’ SAP A N D EVAPORATIXG IT TO CONSISTENCY OF MAPLESIRVP ORGANISM
E . coli A. aerogenes 140 A. a h g e n e s 141 A. aerogenes 142 A . aerogencs 143 11.s. 1 ( Y ) A-4-11,s. 1 B-4-11,S. 1
NORMALITY INCUBAOF ACID TIDN CONSISTENCY OF IN Sap PH PERIOD SIRUP Dam 0.01 5.2 15 Slightly ropy 5.5 0.01 Nearly normal 15 0.01 5 4 Nearly normal 15 0.01 Nearly normal 5.5 15 5 5 0.0i Nearly normal 15 0.005 5.8 Nearly normal l; 0,0125 5 0 Very ropy Very ropy 0.0125 5.0 5
E F F E C T OF N E U T R A L I Z I X Q S.4P J U S T B E F O R E E V h P O R h T I O N
.1-4-M.s. 1
0
. ..
..
S o t quite as ropy a8 the unneutralized
EFFECT O F E V A P O R A T I N G S T E R I L E S A P C O H T A I N I N G ACIDS
ACID Acetic Lactic
COXNSTENCY O F SIRUP A F T E R EYAPN. 0.02 0.015
5.2
4.9
.
..
Normal Slightlyropy
Kone of the known cultures of Escherichia or Adrobactei produced ropiness. There was some change in the concentrated product because of the action of the bacteria on the sap, but nothing approximating the condition found with repTABLE I. ORG.4NISMS ISOLATED FROM s.4P AND FROM PARTI.4LLY resentatives of group I. The changes occurred within 5 BOILED-DOWN SAP INOCULATED INTO 10 PER CENT NORMAL days in the case of the latter bacteria; with the former no MAPLESIRUP change was observed within 15 days. It is interesting that NUMRESULTO F BER DESCRIPTION BOILINQ-DOWN the members of group I produced more acid in dilute maple sirup than did the other organisms tested. M. S. 1 Short small rods, motile in sap; nonmotile Very stringy; strings out when grown on agar Experiments were run to study the effect of acidity on several feet Normal M. S. 2 Very small rods, nonmotile in sap ropiness. Sap that had been inoculated with the bacteria M. S. 4 Nonmotile rod when grown on agar; motile Nearly normal producing ropiness and permitted to ferment for 5 days was in s a p ; very short and small, yellow and neutralized before evaporation, It was then evaporated white on agar to the proper concentration and the degree of ropiness deNearly normal M. S. 5 Nonmotile rods in agar’ some motile in sap, very small and short‘chains termined. There was a noticeable difference in the degree Nearly normal M . S. 6 Nonmotile rods; in agar motile; in sap single of ropiness in it and a sample treated similarly except for and short chains neutralizing the acidity. The neutralized sample was less M. S. 9 Nonmotile, odd-shaped club, rod, and Normal cocci; yeastlike but very small ropy. The two acids, acetic and lactic, which are found in the It is thought that these two differences are not sufficient to gassy fermentation of Escherichia-Aerogenes, were added to warrant naming a new species, especially in view of the well- sterile maple sirup in about the same concentration as was known fact that Aerobacter aerogenes becomes encapsulated produced during the fermentation. The acetic acid had no under certain conditions. As to motility, it has likewise been effect on the ropy condition. The lactic acid had a slight shown that there are certain variants of Aerobacter aerogenes effect (Table 11). that are motile. It is, therefore, evident that this group of LITERATURE CITED bacteria corresponds closely to Aerobacter aerogenes. Edson, Jones, and Carpenter (8) isolated many micro- (1) Bergey, M a n u a l of D e t e r m i n a t i v e Bacteriology, B a l t i m o r e , organisms present in maple sap. Of interest in this connecWilliams and Wilkins Co., 1934. tion is Bacillus aceris Edson and Carpenter, a new species de- (2) E d s o n , H. -4,, Jones, C. H., and C a r p e n t e r , C. W., Vt. d g r . Expt. Sta., Bull. 167 (1912). scribed by them which caused stringiness in the sap, but (3) Stark, C. N., a n d Foster, M. J., Cornell Veterinarian, 21, 109-13 maple sirup from the sap was not especially viscous. They (1931). state that “previous to boiling, the sap was quite stringy, but the resulting sirups were not especially viscid.” Further- RECEIVEDNovember 9, 1934. This paper is Journal Article 193 (N. S.) more a close checking of the characteristics of B. aceris with from the Michigan Agricultural Experiment Station.
