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Surface taint in butter Campbell, Mary Lois 1941

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A is s A ? /5y Surface Taint i n Butter •> 1 1 . • ' ' by Mary Lois Campbell A Thesis submitted i n P a r t i a l Fulfilment of The Requirements f o r the Degree of Master of Science i n Agriculture i n the Department of Dairying The University of B r i t i s h Columbia October, 1941• ^ Contents Introduction • • * Page 1 Part I - The Bacteriology of Surface Taint..... " 3 H i s t o r i c a l . .... «... •« •••••••••«•©.•••••*•• 3 Experimental ....»...«.»•<»««>.••••••••••««* " 10 Part II - Experimental Butters ...... •*..»••••• " 21 Introduction ••••••»***».o.»**.*.******»*» 21 H i s t o r i c a l *«»»«•««..»»»«»•«.«.e.o»»«»e»e® 21 Experimental ......»..»».......••«•••••<>••• ' 25 A. Experimental Churnings (1940) " 26 B. Surface Taint i n Commercial Butters. " 54 C. Experimental Churnings (1941) " VI Part III - The Chemistry of Surface Taint...... " 90 Indole Hypo the sis*.•*.*••.•«•.• • 90 Decarboxylation Hypothesis " 97 Deamination Hypothesis * '• • • ." 99 Summar7/' and Conclusions ....*.*. ••• " 100 Acknowledgements. •.. • ««••«>•••« • • • • • " 107 Introduction. A problem of great importance to the Dairy Industry i s the control of a flavour defect i n butter, commonly known to the trade as Surface Taint. This p a r t i c u l a r type of deterior-ation has been recognized i n Canada since i t was f i r s t described by Marker(31) i n 1^19. It f i r s t demonstrated i t s e l f on the Vancouver Market i n a consignment of F i r s t Grade pasteurized butter shipped from a reputable Alberta Creamery. Although the defect under this name was f i r s t described i n 1919, there i s no doubt but that a condition i d e n t i c a l with or similar to surface taint had been previously observed and described under other names. As f a r back as 1899, butter having a "peculiar f o e t i d odour" was described by Gilruth(17) of New Zealand. Eckles (IS) i n 1900 published a report on butter produced i n Iowa possessing "a disagreeable taste with a putrid smell". In 1910, Orla-Jemsen recorded the occurrence i n Denmark of a butter with "a peculiar putrid odour". The term "rabbito" used to define a defect reported i n A u s t r a l i a i s prob ably synonymous with the term surface t a i n t as employed i n Canada. Other terms have been used to describe the defects and include "cheesy", "limburger", and "surface flavour". The various appelations that have been employed to describe the defect would appear to depend on the stage of i t s development i n butter. The odour and flavour are c h a r a c t e r i s t i c a l l y of a putrefactive nature, although ranc i d i t y quite often develops subsequent to the appearance of the defect. The term "sweaty feet" most nearly defines the odour of surface t a i n t . - .2 -Since the defect was f i r s t recognized i n Canada, the frequent reports of i t s occurrence, p a r t i c u l a r l y i n "butter manufactured i n the P r a i r i e Provinces has made the i d e n t i f i c a -t i o n of the causative agents and control of the defect an xirgent problem. Work on surface taint has been deligently pursued by members of the Di v i s i o n of Dairy Research of the Dominion Department of Agriculture i n co-operation with workers from the Pr o v i n c i a l Universities and Departments of Agriculture. As a large importer of butter from the P r a i r i e Provinces, B r i t i s h Columbia has been much concerned with the question of the control of the defect. The occurrence of surface taint i n butter manufactured i n B r i t i s h Columbia was f i r s t reported i n 1938, and has served to stimulate work on the problem of the cause and control of the defect within the Province. PART I - The Bacteriology of Surface Taint, H i s t o r i c a l Throughout the l i t e r a t u r e on the putrefactive deterior-ation of butter i t i s generally recognized that bacteria play an important role i n the development of the defect* In 1899, Gil r u t h of New Zealand (17) described the de-velopment i n butter of a peculiar f o e t i d odour when held at 65° F» for 36 hours* He attributed the defect to the presence of water bacteria, p a r t i c u l a r l y B a c i l l u s fluorescens liquefaciens e Eckles (12), i n 1900, studied an outbreak of putrid butter i n Iowa c S e v e r a l species of organisms, capable of producing a putrid condition i n butter when inoculated into cream p r i o r to churning, were isolated* One of the species i s described as Bact« fluorescens liquefaciens• Orla-Jensen (28), i n 1910, observed butter possessing a "peculiar f o e t i d odour that ruined i t f o r table use"• Bacteria were shown to be responsible f o r t h i s defect. In work done by Sadler and Vollum (31) mention is made that i n 1919, C. P. Marker, Dairy Commissioner f o r the Province of Alberta, had found deterioration of butter during storage. They state that: "When boxes of the butter were opened, flavour and odour of a most undesirable nature were evinced, the flavour being defined perhaps most clos e l y as "putrid"• The butter was turned out of the boxes, and portions of the outside layer of the respective biocks of butter were removed« Neither flavour nor odour could be detected when the newly cut surfaces were examined* In due course, however, the putrid flavour and un-desirable odour developed i n the butter which had been newly - 4 • -exposed"* •* Sadler and Vollum isolated a number of microorganisms from butter showing this deterioration, and from water and cream at various stages i n the manufacture of butter at the creamery troubled with this problem* A number of their cultures were c l a s s i f i e d as v a r i e t i e s of the species• B, c o l i , B• neopolitanus, B. aerogenes, and B• communior, and other cultures, which they did not attempt to i d e n t i f y were described as Gram negative, motile rods, digesting milk, producing a putrid odour, and f a i l i n g to l i q u e f y g e l a t i n . Three spore-forming strains studied ceased to exhibit the a b i l i t y they possessed of producing gas from glucose, aft e r having been held i n a r t i f i c i a l media for some time* Two of these cultures were e r r a t i c i n t h e i r Gram staining c h a r a c t e r i s t i c s • A neutral c l o t , digestion, and development of a putrid odour were ch a r a c t e r i s t i c of these three cultures. They were isolated from deteriorated butter, cream and water supplies» Spitzer and P a r f i t t (34), In a study of the action of proteol y t i c organisms i n butter, found that cream inoculated with Bo ichthyosmius produced an off flavour and oduur when ripened• Unsalted butter made from cream inoculated with B© ichthyosmius produced an unclean, s t a l e , cheesy odour and f l a v -our after 180 days storage• When the butter was salted, a s t a l e , tallowy and b i t t e r flavour developed. Cheesiness f a i l e d to de-velop i n the case of salted butter. These workers also report-ed the presence of undesirable odours and flavours i n butter made from cream inoculated with B. proteus vulgaris * It was found that of the various type3 studied, the wo ^  ffles p r o t e o l y t i c group of organisms were inh i b i t e d least by an in« creased percentage of s a l t i n the butter© Shutt (33), i n 1929, while examining water supplies of creameries producing surface flavour, found large numbers of putrefactive bacteria, chief of which was Pseudomonas fluorescens« When thi s microorganism was inoculated into s t e r i l e butter and incubated at 25° 0. for 28 days, surface flavour developed. Herreid, Macy and Combs (21) reported that a mixed c u l -ture which produced cheesiness i n butter was found to contain d i s t i n c t species of Achromobacter, Proteus, Streptococus, and Escherichia, and some resembling Salmonella e Hammer and Yale• (18) i n work on the influence of members of the Escherichia en. Aerobacter group of microorganisms on flavour development i n butter, found that butter made from cream inoculated with either Aerobacter oxytocum or Aerobacter cloacae developed off flavours when kept at either 7° or 18° C* No o f f flavours were encountered i n the case of Escherichia c e l l * The defect occurred i n both salted and unsalted butter. The char-acter of these o f f flavours and odours was unclean* resembling somewhat the condition produced i n milk by these organisms• Brown (2) i n 1928, studied the spasmodic occurrence of a defect i n New South Wales butter* The description of the de-fect agrees cl o s e l y with that of surface t a i n t . B a c t e r i a l action was shown to be at l e a s t p a r t l y responsible f o r the de-velopment of the defect* Hood and White (22), i n 1928, i n t h e i r investigation of surface t a i n t butter reported that a large number of l i q u e f y -ing bacteria which decompose the curd of butter were found i n 89 5 8 9 tainted butter* Surface t a i n t was produced experimentally by inoculation of cream with organisms isolated' from creamery water supplies a Derby and Hammer (10), i n 1931, isol a t e d from surface taint butter an organism which was capable of producing the de-fect experimentally. - This organism was te n t a t i v e l y designated as Achromobacter putrefaciens• The f i f t h e d i t ion of Bergey*s Manual of Determinative Bacteriology (1937) does not admit the organism to thi s Genus, but includes i t i n an appendix under a heterogeneous group of organisms, which, according to Bergey, probably should be placed i n some one of the genera included i n the family Pseudoraonadaceae• Long and Hammer (27) have recent-l y designated t h i s organism as Pseudomonas putrefaciens© * Organisms other than Achromobacter putrefaciens, which on inoculation into cream produced 'surface t a i n t i n the butter made therefrom, were also isolated by Derby and Hammer• No c l a s s i f i c a t i o n of these organisms was given© Attempt s were made to produce Surface t a i n t with Ps • fluorescens <, An objectionable condition which may have resembled surface t a i n t appeared for two or three days but ra n c i d i t y soon developed. These workers were not able to produce the defect by inoculating salted or unsalted butter of high quality with surface t a i n t butter. Hussong, Long and Hammer (23), i n 1957, reported that an organism i d e n t i f i e d as Ps• f r a g i i s occasionally responsible f o r cheesiness i n unsalted butter. * Throughout the h i s t o r i c a l sections, the o r i g i n a l genus name i s employed to conform with the name i n the various publications* J . .Campbell (5), i n 1939, isolated a number of organisms from surface t a i n t butter, cream,and water supplies of creamer-ies encountering d i f f i c u l t i e s with surface t a i n t . He was able to reproduce the defect experimentally with a number of these bacteria, especially with two organisms l a t e r c l a s s i f i e d by L. Campbell, as a s t r a i n of Proteus ichthyosmius, and a species of Pseudomonas respectively. Other species isolated were c l a s s i -f i e d as Aerobacter aerogenes, and species of Achromobacter. O u l l i t y and G r i f f i n (8), i n 1938, c i t e d the early work of L o f t u s - H i l l s , Scharp and Searle, of A u s t r a l i a , i n which rabbito i n butter was produced by organisms is o l a t e d from f a c t -ory water supplies, churns, and raw and pasteurized cream. Out-breaks of rabbito investigated by O u l l i t y and G r i f f i n were traced to factory water supplies from which they isol a t e d many types of proteo l y t i c microorganisms• An organism closely resembling Achromobacter putrefaciens was obtained from twowater supplies. In 1939, Wolochow and Thornton (39), postulated that surface t a i n t In butter i s the r e s u l t , not of b a c t e r i a l growth i n or on the butter, but of the presence i n the cream at the time of manufacture "of a product of b a c t e r i a l growth, which i s the precursor of the odoriferous substance recognized as sur-face t a i n t " . They presumed that a chemical change took place i n butter on storage, but stated that the factors influencing t h i s change are unknown* Wolochow was forced to adopt t h i s postulate because he was unable to accept the hypothesis that surface t a i n t arises from the growth of bacteria i n or on the butter, f o r the reason that growth of the causative organism i s in h i b i t e d by a concentration *. 8 -of 4% s a l t . . He argues that those moisture droplets containing higher s a l t concentrations would not support b a c t e r i a l growth, whilst those, of lower concentration would permit development of the organisms with consequent patchy occurrence of the surface t a i n t defect, which, however, i s always uniformly d i s t r i b u t e d throughout a box or churning of butter* The fac t that d i f f i -culty was experienced i n producing the defect by di r e c t inocul-ation of the butter lent support to his thesis* In studies on the influence of working on commercial churnings, Woloxshow and Thornton (39) found that the defect was confined to unworked samples and offered as an explanation of this finding that surface t a i n t i n the unworked butters was pro-duced by b a c t e r i a l growth i n the unincorporated moisture of these butters, and that the fi n i s h e d butters were protected from such growth because of the fin e moisture dispersion obtained as a re-sult of working. The basis f o r t h i s reasoning depends not only upon the s a l t concentration of the dispersed waterdroplets i n -h i b i t i n g growth, but as well on the size of the droplets f a i l -ing to permit b a c t e r i a l m u l t i p l i c a t i o n . Further, they were led to conclude that the agent responsible f o r surface t a i n t was widespread* In a l a t e r study, Wolochow and Thornton (40) reported i d e n t i c a l findings, but placed d i f f e r e n t i nterpretation on the results • Failure to isol a t e A!chromobacter putrefaciens, the causative Agent, from many of the alleged surface t a i n t butter' samples, led them to question the authenticity of t h e i r f i n d -ings • They state that "Putrefactive bacteria of many types probably grow i n the free moisture of the unworked butters", and that "the odours a r i s i n g from these butters could e a s i l y be mistaken for the S. T. odour", and that "the results had questionable bearing on the surface t a i n t problem"• While Derby and Hammer (10) reported that several types of microorganisms were capable of experimentally producing sur-face t a i n t , t h e i r work indicates that the p r i n c i p l e microorgan-isms responsible for the defect i s Achromobacter putrefaciens• They showed c l e a r l y that special procedures are required for i s o l a t i o n of this species and suggested that f a i l u r e to is o l a t e Achromobacter putrefaciens from surface t a i n t butter was i n a large measure due to d i f f i c u l t i e s associated with the technique, of i s o l a t i o n . The procedure recommended by Derby and Hammer i s en-richment i n litmus milk, holding at 5* C. u n t i l reduction occurred, followed by plating on beef infusion agar. Claydon and Hammer (6) suggest other procedures for the i s o l a t i o n of Achromobacter putrefaciens. Pasteurized cream i s inoculated with the o r i g i n a l defective butter and incubated overnight at 10° C. It i s then churned, and the resu l t i n g un-salted butter i s divided and held at 21" C. .and 5° C. u n t i l the de-fect i s reproduced. When the butter has become putrid, i t i s plated and the colonies picked into litmus milk. Another i s o l -ation method suggested by these workers i s a modification of the Bur r i Smear Culture Technique. Instead of Agar slants, plates of beef infusion agar plus skim milk f a t emulsion are poured and allowed to s o l i d i f y . The plates are marked into s i x sectors, and each sector smeared with a t i n y portion of butter. These plates are incubated at room temperatures f o r f i v e days. Col-onies are thus e a s i l y recognized and picked. -.10-l n 1941, Long and Hammer (27) evolved a medium more spe-c i f i c for the i s o l a t i o n of Pseudoraonas putrefaciens• This med-ium i s a special F e r r i c ammonium c i t r a t e g e l a t i n agar* On t h i s medium Ps. putrefaciens appears as a brown to brownish red or pink colony. They employed the di r e c t smear technique as well as enrichment i n litmus.milk p r i o r to plating* Using t h i s pro-cedure, Long and Hammer showed that Ps. putrefaciens i s widely dis t r i b u t e d , not only in' dairy products (milk, cream, and butter) but as well i n water, s o i l and creamery equipment. In 1940, Wolochow and Thornton (40) described an i n t e r -esting test f o r the detection of Achromobacter putrefaciens when grown i n s t e r i l e skim milk. This organism was found to produce a cha r a c t e r i s t i c odour with properties which have not been dup-l i c a t e d by any other organism studied by them. The odour i s des-cribed as that of "sweaty feet" and has been shown to possess the property of becoming i n t e n s i f i e d when the milk culture i s spread between the fingers and allowed to dry. Raw milk inoculated with Ach, putrefaciens f a i l s to y i e l d the c h a r a c t e r i s t i c sweaty feet odour• Experimental, The work reported upon i n Part I of this thesis describes i n d e t a i l , the c u l t u r a l characteristics of the microorganisms employed throughout the study, and includes a description of a suggested procedure for t h e i r detection and i s o l a t i o n . Pour of the cultures were o r i g i n a l l y i s o l a t e d by Campbell (3) i n studies on surface t a i n t i n butter, and were l a t e r c l a s s -i f i e d by the author (5) as species of Pseudomonas, Achromobacter, Proteus, and Aerobacter, Three microorganisms were is o l a t e d during the course of this study and t e n t a t i v e l y c l a s s i f i e d •» 11 «• within theA Genus Pseudomonas• Two cultures, Pseudomonas putre-faciens and Proteus ichthyosmius, o r i g i n a l l y i s o l a t e d by Hammer were obtained through the courtesy of Professor Hammer• A detailed description of the c u l t u r a l and biochemical character-i s t i c s of these microorganisms i s given below: --Pseudomonas Ptitrefaciens « Morphology Rods; o*55 x 2.1 u; singly, i n pairs, and short chains; Gram negative; Motile by means of a polar flagellum; (confirmed by author). ' ' Cultural Characteristics Beef Infusion Agar Convex, g l i s t e n i n g , smooth, trans-lucent, colorless to grayish colony becoming brown to reddish brown on aging. Special Gelatin Agar - Colony characteristics similar with exception of colour - brown to red-dish brown or pink; putrid odour. Gelatin Stab — — Crateriform l i q u e f a c t i o n . Beef Extract Broth — Turbidity, sediment, and t h i n p e l l -i c l e . Litmus Milk — R a p i d l y reduced, usually 6-8 hours, by active cultures; proteolysis• Biochemical Ch a r a c t e r i s t i c s . Indole .----•..»-».....--...».. Not produced Nitrates — — Reduced to n i t r i t e s H2S — Positive M. R. — — - — — — » Negative - 12 -V. P. — — --—.Negative — Positive Fermenting Power — - — Some strains produce no change i n any of the various boiullons, while others produce acid but no gas from maltose, sucrose, arabinose, dextrose, galactose, lactose, and levulose. L i p o l y s i s ---------- Negative Phosphatase •—.— Rapidly produced i n milk (not con-firmed) Growth Conditions Facultative; grows at 3* - 30 C ; grows i n 4$ NaCl; some strains i n 6 - B% NaCl. Proteus ichthyosmius (Hammer) Morphology Rods; 0,6 to 0,8 u x 1.0 to 2.1 u; singly; Gram negative; motile by mean of peritrichous f l a g e l l a . (Confirmed by author) Cultural Characteristics. Agar Small, white colonies, darkening with age. Broth — T u r b i d with gray sediment. Gelatin Stab- Liquefaction. Litmus Milk - Acid; reduced. Cultures have f i s h y odour. Biochemical C h a r a c t e r i s t i c s . Indole — - - - Produced (Confirmed by author) Nitrates — — Reduced to n i t r i t e s . M. R. — Positive V. P. ——>—-- Negative. - 13 -Fermenting A b i l i t y — Produces "acid and gas from dextrose, levulose, galactose, maltose, sucrose, g l y c e r o l , s a l i c l n , and mannitol. Growth Conditions Facultative; opt. temperature 30 C. Proteus ichthyosmius (Campbell) Morphology Rods; .8 x 1.6 u; square ends; s i n g l y sand i n pairs; Gram negative; Motile by means of peritrichous f l a g e l l a . Cultural Characteristics Standard Agar (Tryptone Glucose Extract Agar) —-• Pro t e o l y t i c , small white, c i r c u l a r colon-ies . Davis Broth — - Slimy p e l l i c l e ; dense growth with slimy sediment. Gelatin Stab — Complete l i q u i f a c t i o n . Litmus Milk — Firm, acid c l o t ; reduction i n 2 days with marked p r o t e o l y s i s . Biochemical Characteristics Indole Not produced Nitrates — Reduced to n i t r i t e s . H 2S « - — — — Positive NHg Positive M.R. — — — — — Negative V.P. — Positive Fermenting Power Acid and gas i n sorbite, mannite, glucose, sucrose, and s a l i c l n ; gas -• 14 -••„• , but only s l i g h t acid i n glucose and maltose* L i p o l y s i s Positive Phosphotase — • — Negative Growth Conditions Facultative; grows at 23°•• - 47°C; opt* 30° G; grows i n 6% NaCl. Aerobacter .aerogenes Morphology Rods; .6 x .7 u; square ends; singly and i n pairs; Gram negative; Motile by means of one or two polar f l a g e l l a . Cultural Characteristics Standard Agar — Proteolytic; smooth, white colonies. Davis Broth — Gas produced; dense growth; heavy sediment. Gelatin Stab — • •— No li q u e f a c t i o n Litmus Milk — — --Reduced, hard gassy c l o t ; surface proteolysis• Biochemical Characteristics. Indole * — N o t produced Nitrates — - - - - — — Reduced to n i t r i t e s • H g S - - — - - - - - — — Positive NHg — Positive M. R. — N e g a t i v e V. P. r — Positive Fermenting A b i l i t y - Produces acid and gas from sorbite, mannite, glucose, sucrose and lactos - 15 -Lip.olysis ----- .- Negative© Growth Conditions . Facultative; grows from 23°C. - 37° C.; grows i n 6% NaCl, Achromobacter»(Species name not given - unlike any species . , described i n Bergey (1).) Morphology Rods; .8 x .9 u; rounded ends; singly and i n pairs; Gram negative; motile by means of a single polar f l age Hum. Cultural Characteristics Standard Agar Proteolytic; yellowish colonies. . Davis Broth Dense growth; gummy sediment Gelatin Stab -—•— Complete l i q u i f a c t i o n Litmus Milk Proteolysis Biochemical Characteristics Indole • Not Produced Nitrates • — Reduced to n i t r i t e s HgS • Positive HH3 ~—•• — Po s i t i v e M. R. •-— — ---- Negative V. P. •-»- Negative Fermenting A b i l i t y Produces acid i n glucose L i p o l y s i s • Positive Growth Conditions, F a l c u l t a t l v e ; opt. temperature 23° ; grows i n 4% NaCl. Pseudomonas(Species name not given - unlike any species described i n Bergey) Morphology Rods; .6 x .8 u; singly,-and i n pairs, and short chains; : Gram negative; Motile by means of a single polar flagellunu Cultural Characteristics Standard Agar — P r o t e o l y t i c , smooth f l a t colonies with a greenish fluorescence. Davis Broth — — — Dense growth; gummy sediment; f l u o r -escence at 5° - 30° G. Gelatin Stab ----— Complete Liquefaction Litmus Milk Alkaline; reduced; complete proteolysis fluorescence. Biochemical Characteristics Indole — — • Not produced Nitrates — — • - . •- Not reduced HgS — Negative NH ——. Positive Mf.R. — — " Negative. V.P. — Negative Fermenting A b i l i t y - Produces some acid i n glucose,, L i p o l y s i s - — Positive Growth Conditions Facultative; grows at 5° - 47° C ; opt. temperature 23° 0.; grows i n 6% NaCl. Cultures B 9 and B 13 — to be c l a s s i f i e d as strains of P'S« putrefaciens on t h e basis • °f ld.ehti£.i;-fcat--lo&: procedure , , • . proposea.-by Hammer (27). ' Morphology Rods; gram negative• fa?yptie Casein Digest Agar (Ferric Amm. c i t r a t e and Gelatin added) Pink, mucoid, g l i s t e n i n g , convex colony. Litmus Milk Rapidly reduced - 17 -Nitrates Reduced to n i t r i t e s . Fermenting A b i l i t y - Do not ferment glucose or lactose. Odour produced i n Butter --• Surface Taint. Isolation Technique for Surface Taint producing Bacteria. The d i f f i c u l t y with which surface t a i n t producing bact-e r i a i n i t i a t e growth on laboratory media, has been responsible i n large measure for the slow progress i n determining the f a c t -ors essential to the development of surface taint i n butter* From time to time various changes i n procedure for the i s o l a t i o n of the causative microorganisms have been proposed, involving alterations i n the type of medium and the method of p l a t i n g . In studies on the nature of the medium required for the growth of surface t a i n t producing organisms, the majority of the work reported herein was carried out employing Ps. putrefaciens, the most fastidious of the organisms i n so far as i n i t i a t i o n of growth i s concerned. Repetition of Hammer's work (25) con-firmed his finding that the modified B u r r i Smear Culture Tech-nique was superior to the d i l u t i o n method of platings In preparing the special Iron Gelatin Agar described by Hammer, considerable d i f f i c u l t y was encountered i n obtaining a s a t i s f a c t o r i l y clear medium. I t was l a t e r found that the source of t h i s d i f f i c u l t y was the peptone employed as the nitrogen source. As the work progressed, various types of nitrogen source were employed, including Peptic Casein Digest Broth af t e r Orla-Jensen (29), and Tryptic Casein Digest Broth after Cole (7). For purposes of i s o l a t i o n and detection of Ps. putrefaciens, the agar containing Tryptic Casein Digest Broth was found superior as a medium to the o r i g i n a l agar described by Hammer, i n which . . . • - 18. -proteose peptone served as the source of nitrogen. On this medium i n i t i a t i o n of growth of cultures of Ps. putrefaciens, employing either the direct plating or the modi-f i e d Burri Smear techniques, was much faster and the growth was markedly more luxuriant• This medium has been employed through-out the greater part of the work reported upon i n t h i s thesis as a medium for the i s o l a t i o n of surface t a i n t producing bacteria from butter, creamery water supplies, and other sources« .The Detection of Surface Taint Producing Bacteria i n Milk. The work reported upon by Wolochow and Thornton (40) i n which an interesting test f o r the detection of Ps. putrefaciens involving the recognition of a characteristic odour produced by the culture i n pasteurized skim milk, suggested that a study of the odour producing a b i l i t i e s of the various types of micro-organisms used i n t h i s investigation and capable of producing surface t a i n t i n butter, should be undertaken. For the purpose of t h i s series of experiments, raw milk of high quality was obtained 6 It was divided into four equal portions. One portion was dispensed i n test tubes i n 10cc© quantities, and autoclaved at 245° F. f o r 10 minutes, another portion tubed i n the same manner and autoclaved at this tem-perature for 25 minutes; a t h i r d portion was placed i n s t e r i l e test tubes and pasteurized at 145° F. for 30 minutes, and the remaining portion was tubed s i m i l a r l y and received no heat treatment. These four milks were then inoculated with the following organisms: Ps * putrefaciens (Hammer), P rot. i c h -thyosmius (Campbell), Aerobacter aerogenes, and species of Pseudomonas and-Achromobacter, and incubated for 9 days at 23° C. At the end of t h i s time, the tubes were shaken, three or four drops taken between thumb and finger, and rubbed u n t i l the moisture had evaporated, observing the resulting odour mean-while. The findings are detailed i n Table 1. Consideration of the results shows that with the major-i t y of the microorganisms, the most undesirable odours occur i n the case of those cultures inoculated into pasteurized milk© Some odours did develop i n certain of the raw and autoclaved samples, but they were much less intense. Ps• putrefaciens pro-duced the cha r a c t e r i s t i c "sweaty feet" odour, Prot. ichthyosmius (Hammer) a d e f i n i t e f i s h y smell, and Prot. ichthyosmius (Campbell) a combination of the sweaty feet and f i s h y odours. The other organisms produced less powerful odours, sweaty feet i n the case of the Psdudomonas species and f i s h y and putrid i n the case of r. the Achromobacter species. -20-i " FT"— T 1 •d 1 43 I 1 <M •ri i 1 CO 1 1 © o •ri | •P 05 ft 0 © O © >s h> •ri U cd H H +3 ft; •P ««• -P s a CO |»s 8 43 S» 43 s a © +3 8 W 43 W a bo oJ 1 •ri CO •ri bO © H •ri H d £. co ft CO COCO •ri •d S •ri -P <ti O 'd 43 to «»« •ri d •d •P -p O 43 ft © & © © © NO © © •ri «H s>> q l>> H o! l>> +3 o! <D ft •P b -P \>> 43 • 43 43 05 43 W 43 w to CD CO © co •ri © CO & •ri £ «ri H •ri H ft CO ft CO CO ft S •ri S 8 •ri .43 43 Ctf 03 is CO •ri •XJ k ft <M <D O ft <M > <H !>> s 1 <!*! J c! 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I < - 21 * •- PART II - Experimental Butters , Introduction The sporadic occurrence of surface t a i n t i n the manu-facture of butter commercially has made d i f f i c u l t the exact de-f i n i n g of the conditions governing i t s development e Although i t i s generally agreed that the occurrence of surface taint i n high grade butter made from pasteurized cream i s due to the a c t i v i t y of microorganisms, the microbiological deterioration of butter i s influenced by other factors entering into the manufacturing process* The a c i d i t y of the cream, the extent and method of neutralization, the degree of working and i n -corporation of moisture, and the concentration of s a l t are factors which have been shown to be of importance i n the pro-duction of the defect experimentally* H i s t o r i c a l * Derby and Hammer (10) showed that surface t a i n t was more r e a d i l y produced i n butter made from cream of low a c i d i t y than from cream of high a c i d i t y . A high percentage of s a l t i n butter usually prevented the development of the de-f e c t . They state that "the prominence of surface t a i n t i n re-cent years i s probably due the great changes that have been introduced i n the methods of the manufacture of butter". They refer s p e c i f i c a l l y to the improvement i n the quality of cream used for butter manufacturing, and to the public demand for butter of low s a l t concentration. Hood and White (22), i n 1928, found that many samples of surface t a i n t butter come within, the same a c i d i t y range as that of normal butter. In studies on the eff e c t of neutral-22 i z a t i o n on the development of the defect, these workers found that overneutralization or careless methods of neutralization were not responsible for i t s occurrence. They concluded from the i r work on the in h i b i t o r y e f f e c t of s a l t that "the question of "controlling the undesirable organisms by high s a l t i n g i s im-practicable i n view of the fact that the market for western butter demands a butter of s a l t content lower than 2•67$"• Sproule and Hamilton (35), i n 1937, i n studies on surface t a i n t i n Ontario butter suggest that possible reasons for the increased incidence of the defect are: (1) an increase i n the percentage of butter made from high qual-i t y cream of low a c i d i t y . (2) the increasing tendency towards the manufacture of butter of low s a l t content. Wolochow and Thornton (40) found that the l i m i t i n g a c i d i t y for the growth of Ach. putrefaciens i n cream was approx-imately pH 5.6, roughly corresponding to a t i t r a t a b l e a c i d i t y of 0.3$. They also found that Ach. putrefaciens produced sur-face t a i n t -in pasteurized cream with a s a l t content up to 2,5%, Campbell (3), i n 1939, found that overneutralization of the cream appeared to have no ef f e c t , either stimulatory or in h i b i t o r y , on the development of surface t a i n t . A low temper-ature of neutralization inhibited the development of the defect. In l a t e r work Campbell (4) reported that the optimum t i t r a t a b l e a c i d i t y of cream for the production of surface t a i n t i n butter was .11 - .18$ t i t r a t a b l e a c i d i t y . Derby and Hammer (10) were able to reproduce surface t a i n t when experimental butters made from cream Inoculated with the s p e c i f i c organisms were incubated at 15.6 ° G. (60°P.) f o r 2 to 4 days, and at 5*0 (41 P.) for 7 to 10 days. Hood and White (22) state that surface t a i n t begins to appear i n butter eight to ten days after manufacture; the degree of development o the defect depends somewhat on the temperature of previous storage, and more rapid development takes place around 40° to 45°F. Wolochow and Thornton (39) reported the development of ' surface t a i n t i n butter stored as low as - 5°C. Derby and Hammer (10) i n making experimental butter, infected the butter by inoculating the s p e c i f i c organism into the cream prior to churning. They were unable to obtain the defect by inoculating normal butter. Campbell (3) was able to reproduce surface t a i n t experimentally by smearing cultures of organisms responsible for the defect on the surface of normal butter* Sadler and Vollum (31) i n th e i r studies on deterior-ation of butter, inferred from their results that i n order to secure a high grade butter, every e f f o r t should be made to se-cure i n i t i a l cream of the highest quality; cream i n which the b a c t e r i a l f l o r a present has had but l i t t l e opportunity to pro-duce a c i d i t y or other flavours. Campbell (4) showed that the incidence of surface t a i n t i n butter made from cream i n which the growth of a certain organism had occurred prior to pasteur-i z a t i o n and subsequent i n f e c t i o n with a surface t a i n t producing organism than i n butter made from pasteurized cream to which t h i "associated" microorganism had not been added. He found that E. neopolitana, Aer. cloacae, Aer. aerogenes, and E. c o l i were p a r t i c u l a r l y e f f e c t i v e i n bringing about this precondition for the development of the defect• 24 -Long and Hammer (24) i n studies on the influence of working on the dispersion of moisture and i t s effect on defects i n butter showed that bacteria i n butter are large l y contained i n the water droplets, and that their growth i s r e s t r i c t e d In large measure to these infected droplets. In underworked butter the droplets are r e l a t i v e l y large. In thoroughly worked butter they are small and well separated by f a t . A decrease i n size of the water droplets results i n a lessened supply of nutrients available f o r b a c t e r i a l growth. These workers were led to con-clude that "the d i s t r i b u t i o n of moisture i n butter i s a factor -tending to influence the growth of bacteria, and changes due to ba c t e r i a l action should take place more slowly i n thoroughly worked than i n under worked butter "• Because organisms are more active i n underworked than i n well worked butter, they suggest adequate working as a means of protecting butter against certain defects, including surface t a i n t . Long and Hammer (26) i n further studies on defects i n butter found that reworking of unsalted butter made from pasteur-ized cream inoculated with various organisms, and held at 10*C, usually resulted i n an increased b a c t e r i a l growth, and a decrease i n the time required f o r the development of defects• They also found that the printing of butter i n a type of equipment which subjected i t to a reworking process tended to aggregate the moisture droplets. The contamination of previous-l y uninfected water droplets and the provision of a greater food supply by aggregation of the moisture droplets were shown to be important factors i n the acceleration of various microbiological changes.that take place i n butter on reworking© Long and Hammer - 25 -further found that reworking of butter after several days stor-age commonly resulted i n a more rapid and extensive increase i n the number of bacteria, than i n butter reworked shortly a f t e r manufacture. Defects were also found to develop faster i n but-ter' that had been held longer p r i o r to reworking, Achromobacter putrefaciens was used as one of the organisms i n these studies© Claydon and Hammer (6) were able to develop surface t a i n t experimentally with Ach, putrefaciens i n one day at 21° C., and i n seven days or l e s s at 5°C« They found that salt,, while tending to i n h i b i t this defect was not completely e f f e c t i v e un-less the butter had been thoroughly worked, Expe r imental 6 The studies reported upon herein were undertaken with the object of determining the conditions governing the develop-ment of surface t a i n t i n butter. Consideration was given to the following factors: 1, Type of microorganism, 2, Associative b a c t e r i a l action, 3, Mode of i n f e c t i o n , 4, A c i d i t y of cream, 5, Neutralization, 6» Degree of working, 7« Salt percentage, 8, Reworking, 9, Storage conditions, including temperature and aeration. - 26 -fAKT XI A - Experimental Churnings» Procedure and Methods . For the experimental churnings, 6-7 pounds of raw sweet cream were standarized to 33$ butterfat,pasteurized at 175°F« (79 C.) for ten minutes, cooled immediately and held overnight at 4° C. J u s t p r i o r to churning, a c i d i t y determin-ations were made© The cream was churned i n a No• 5 Buttercup stainless s t e e l hand churn, s t e r i l i z e d by autoclaving•• A churning tem-perature of approximately 44 - 46° F.* employed. A control sample was taken d i r e c t l y from the churn after the buttermilk had been drained off p r i o r to washing. The butter was then washedtwice with s t e r i l e wash water, which had been inoculated with approximately .033$ of a young Davis (9) Broth culture of the microorganism understudy. Salt-was added at the rate of •§• - % oz • per pound of butter. The butter was worked on a small wooden hand worker s t e r i l i z e d by chemical treatment. Butter samples were taken a s e p t i c a l l y i n s t e r i l e 5" glass bottles equipped with a i r - t i g h t aluminum screw caps• The organisms employed i n this pj. r t of the work were representative cultures i s o l a t e d from surface t a i n t butter, cream, or creamery water supplies by Campbell (3). They i n -cluded a s t r a i n of Proteus ichthyosmius, Aerobacter aerogenes, a species of Pseudomonas, and a species of Achromobacter• Pseudomonas putrefaciens, obtained from Professor Hammer was also employed. The b a c t e r i o l o g i c a l examination of the butters was made by the plate method, using Standard Agar (Tryptone Glucose - 27 -Extract Agar)* Samples were plated the day of manufacture, and after one week's storage* The pH of the f i n i s h e d butter serum,obtained by melt-ing the butter, was determined with a quinhydrone potentiometer. The butter samples were graded at suitable intervals by Mr© H« A. Mason, Dominion Dairy Producer Grader for the Prov-ince of B r i t i s h Columbia* Ef f e c t of Degree of Working and Temperature of Storage on Development of the Defect. Long and Hammer maintain that the incorporation and dispersion of moisture are important factors influencing the b a c t e r i a l deterioration of butter, and have a d i r e c t bearing on the development of defects. In order to determine the d i r e c t influence of this factor on the development of surface t a i n t , samples were there-fore taken at the following stages of working. " I . Very s l i g h t l y worked. I I . Worked s l i g h t l y more. I l l • S l i g h t l y underworked. IV. S u f f i c i e n t l y worked. V<® Extremely overworked. In order to s tudy the effect of temperature on the a c t i v i t y of the microorganism i n butter worked to varying de-grees , duplicate samples of each stage of working were taken. One sample was s tored under conditions very favourable for the growth of the microorganism - 23°C. (73.4°F.), while the other sample was held at approximately 5°C. (41 F.) for one week and then transferred to 23°C. p r i o r to examination. - 28 * i Discussion. The findings are recorded graphically i n Figures 1 to 21 i n which the logarithms of b a c t e r i a l counts are plotted a-gainst degrees of working* Although the peculiar odour and flavour character-i s t i c of surface t a i n t was r a r e l y encountered, other equally undesirable odours and flavours were consistently obtained. These were variously described as "Nearly S.T.", "putrid", " d i r t y " , "cheesy", "amine", and "synthetic". The word "synthetic 1 was used to describe the p a r t i c u l a r odour which suggested the smell produced by various combinations of synthetic amines i n butter, as reported by Campbell (3), There is no sharp di v i d i n g l i n e between these cl o s e l y related types of odours, since, l i k e other b a c t e r i a l defects, each undergoes a sequence of changes dependent upon microbiolog-i c a l a c t i v i t y . One defect may predominate and be replaced by another, or a blending may r e s u l t i n a defect suggestive of more than one type 8 These undesirable defects occurred more frequently i n underworked butter than i n well worked butter. When the defect appeared i n a l l or i n the majority of the butters at the various stages of working, there was usually a decrease i n potency of the odour with an increase i n the degree of working. F i g s . 3,5,6,9, XX j X2 $ X4 £ X 5 e Because the incidence of ch a r a c t e r i s t i c surface t a i n t i s low i n t h i s series of experiments, nothing d e f i n i t e can be said with regard to the influence of temperature of storage on i t s development. However, the production of closely related un-— 29 — desirable odours occurred more often i n the butter samples that were stored one week at 4* C , and then held at 23°C, for a short period of time, than i n those that were kept at 23°C. throughout the holding period. Pig, 1, 2, 11, 12, 13, 14, 15, Undesirable odours were more often encountered i n experimental butters inoculated with cultures of Prot, Ichthyos-mius (Campbell) and Pseudomonas (Campbell) than when cultures of Aerobacter aerogenes and Achromobacter (Campbell) were em-ployed. F i g , 1 - 4 , Fi g s , 1 - 2 1 show c l e a r l y the influence of the degree of working and the temperature of storage on the growth of microorganisms i n butter. In the case of the b a c t e r i o l o g i c a l examinations made on the day of manufacture, a tendency towards a d e f i n i t e decrease i n b a c t e r i a l count as the extent of working increases i s to be seen, F i g , 1 - 21, The b a c t e r i a l counts on the samples held at 5°C, for one week show that for the lesser degrees of working, there has been considerable microbial m u l t i p l i c a t i o n i n the case of certain of the experimental churnings• The majority of the samples showed l i t t l e evidence of change, whilst i n others the b a c t e r i a l count was actually lower after one week's storage than on the day of manufacture• Many of these samples were conspicuously tainted, As the de-gree of working increased, the b a c t e r i a l counts of the samples stored at 5°C, showed i n general a marked decline approximating the count obtained on the o r i g i n a l "extremely overworked" butter, Figs. 1, 3, 4, 6} 0 20. When the butters were stored at 23*C., a marked i n -crease i n count i s to be seen i n the case of p r a c t i c a l l y a l l — 30 — samples, Pigs• 1 - 9, 10 - 18, 20. In the samples submitted to " s l i g h t working", the r e l a t i v e increase i n numbers i s higher than i n the samples worked to a greater extent. In most cases, even when held at 23*0•, the "extremely overworked" samples showed but a s l i g h t increase i n b a c t e r i a l count,, Associative Action of Microorganisms. In work on the influence of the associative action of other organisms on the subsequent a c t i v i t y of surface t a i n t bacteria, the buttermaking procedure was altered to the extent that the raw sweet cream was inoculated with the "associated" bacterium, Incubated overnight, and neutralized p r i o r to pas-teu r i z a t i o n , o,025$ Inoculum of a young Davis Broth culture of the "associated" organism was employed. Sodium carbonate or bicarbonate was used as the neutralizing agent• The butter was made after the manner described above, employing the wash water as the channel of i n f e c t i o n for the surface t a i n t organism. As the work progressed, a further change i n technique was adopted. At the fourteenth churning, the amount of s a l t used wasrreduced to one t h i r d of the amount previously employed. This departure from the previous procedure was adopted as a res u l t of the f a i l -ure to consistently reproduce the c h a r a c t e r i s t i c surface t a i n t defect, and i n the l i g h t of results reported by other workers (10), (22), (35). Discussion, In churnings 5 - 20, the influence of the .growth of associated microorganisms with the resultant changes i n the a c i d i t y of the cream and the neutralization problem thus created along with other petit inent factors to which reference has already been made, have been studied. - 31 -Although the characteristic surface t a i n t defect was not encountered i n the experimental churnings, a higher i n c i d -ence of undesirable odours and flavours was found i n the case of these butters than i n the series i n which only surface t a i n t producing organisms were employed* Subsequent to the reductionin the salt content of the experimental butters, defects closely akin to surface t a i n t were more commonly encountered* This plate 10 x 7 inches. Edge of each small square 1/10 inch. TV 33 -This plate 10 x 7 inches. Edge of each small square 1/10 inch. COO T. This plate 10 x 7 inches. Edge of each small square 1/10 inch. CCC. This plate 10 x 7 inches. 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Edge of each small square 1/10 inch. c c.c. T. This plate 10 x 7 inches. Edge of each small square 1/10 inch C C . C . T This plate JO x 7 inches. Edge of each small square J/IO inch. ccc T. .ao,.i. i t 4 I- rt:, Organisms' laJlftl f. 1-^.peTiidbmpnas, , , i;( bampTDelli) md .; I , ,_4 L "OB V In. J _1- 1-(-Campbell -I J I j, .iTJ This plate 10 x 7 inche.. Edge of each small rquarc 1/10 inch c c.c. T, This plate 10 x 7 inches. Edge of each small square 1/10 inch. C C.C. T-~ 54 -- . PART II B - Surface Taint In Commercial Butte It has been reported by marketing organizations that In certain instances, butter i n prints has developed surface t a i n t , while butter from the same ehurnlng, but marketed i n boxes has f a i l e d to evidence the defect. The f a c t that there seems to be a relationship between the type of butter printer used and the development of surface taint strongly suggests that this type of printer may be more d i f f i c u l t to maintain i n good sanitary con-d i t i o n , or that a change of moisture d i s t r i b u t i o n brought about by the action of t h i s form of equipment i s the factor mainly responsible for the development of the defect• The degree of aeration to which the butter i s subjected by this process may also contribute to i t s production. These changes induced i n the physical nature of the butter by the reworking process may be responsible for an i n -creased b a c t e r i a l growth res u l t i n g i n the development of sur-face t a i n t . The question of the temperature of the butter i n i t s r e l a t i o n to these alterations i n physical structure, and t h e i r influence on b a c t e r i a l a c t i v i t y i s undoubtedly of para-mount importance• In order to determine the s p e c i f i c effect of each of these factors and t h e i r relationship one to the other on the development of surface taint i n butter, the following series of experiments were performed. Experiment I» For the purpose of t h i s experiment several pounds of F i r s t Grade Creamery butter taken from a 56 pound box were ob-tained. Using aseptic precautions, the block of butter was 55 divided into two equal sized parts, confining the entire ex-posed surfaces to one portion* These portions are designated as "Surface" and " i n t e r i o r " . Each of these portions was then divided into two parts. One of each of the Surface and Inter-i o r portions was then put through a s t e r i l e meat grinder, sim-ulating, the conditions encountered at times i n the commercial pri n t i n g of butter® Samples of the reworked butter, designated as Surface Grinder and Interior Grinder were then taken. The remaining half of each portion was sampled d i r e c t l y . In a l l , nine samples of each of the four.portions - Surface, Surface Grinder, Interior and I n t e r i o r Grinder - were taken, employing s t e r i l e t r i e r s . One sample of each portion was plated immed-i a t e l y on Standard and Malt Agar, The method adopted f o r the storage of the remaining samples was designed with the object of providing varying de-grees of oxygen supply at d i f f e r e n t temperatures. A v a r i a t i o n i n the oxygen supply was obtained by preparing samples i n the following manner: 1o Containers f i l l e d with packed butter© 2 o Containers h a l f - f i l l e d with packed butter« 3, Containers h a l f - f i l l e d with unpacked butter, 4, Containers h a l f - f i l l e d with packed butter and deprived of oxygen by placing i n an oxygen free atmosphere• Pyrogallic acid was employed for t h i s purpose. Duplicate samples of each variation were prepared, one sample being held at 5°C. and the other at 23°C. for a period of seven days • They were then plated on Standard Agar and on Malt Agar, The counts obtained are recorded i n Table 2, Duplicate samples -•*> one placed at 5 C., the other at 23 C. Discussion The r e s u l t s recorded i n Table 2 fo r the butters p l a t -ed Immediately aft e r sampling show c l e a r l y that the butter em-ployed was of low b a c t e r i a l contents The figures for the t o t a l counts on the Surface and Interi o r samples indicate that the d i s t r i b u t i o n of bacteria In butter i s not uniform and confirm the findings reported by Hammer (10)• The t o t a l counts ob-tained on the "Surface", and "Surface Grinder" samples show that the methods used i n the sampling and handling of butter for t o t a l b a c t e r i a l count determinations may be of paramount Im-portance • The only difference between the two samples i s that p r i o r to plating , the Surface Grinder sample had been subject-ed to a grinding process. The explanation of the phenomenon may be simply that t h i s process results i n a wide d i s t r i b u t i o n of organisms throughout the butter, re s u l t i n g i n an increased count on the aliquot taken for b a c t e r i o l o g i c a l analysis i n the case of the Surface Grinder sample. The increase i n count, however, may. only be an apparent one, the lower count obtained on the butter not exposed to grinding being due to the pecul-i a r i t i e s i n the growth requirements of the bacteria present i n » 5 8 " the butter, with resultant f a i l u r e to develop colonies on plates The f a i l u r e to i n i t i a t e growth on media i n the case of the un~ ground butter may be dependent upon the extent of aeration to which the butter i s subjected during the grinding process. The grinding process appears to be without influence on butter taken from the i n t e r i o r oS the block so f a r as the number of bacteria, as revealed by the Plate Method, are con-cerned. Table 2. However, the d i s t r i b u t i o n of bacteria with .respect to species would appear to be altered by the process*, Proteolytic types constituted a much larger proportion of the t o t a l numbers of bacteria appearing on Standard Agar plates. This finding i s also to be seen when the results of the Surface and Surface Grinder samples are observed. Although the Surface sample showed no proteolytic types, these made up one quarter of the t o t a l count of the Surface Grinder butter* The influence of the simple process of grinding on the micro-biological analysis of butter Is s t r i k i n g l y revealed when the yeast and mould counts on the four butter samples are considered, Table 2• The grinding of butter, whether from the surface or the i n t e r i o r of a block, results i n a marked increase i n yeast and mould count. Whatever the explanation of this phen-omenon may prove to be, the finding i s of significance i n so f a r the determination of the yeast and mould count i n butter i s con-cerned • This method i s an accepted standard for the evaluation of butter q u a l i t y . The findings reported herein indicate that further study should be made of the methods used for the taking and preparing of s amples for yeast and mould count determinations Although errors inherent i n the Plate count of butter ~ 59; - ' make d i f f i c u l t the interpretation of resu l t s , the findings re-corded, i n Table I, show c l e a r l y the influence of the grinding process on the b a c t e r i a l population of butters stored at 5°C and 23°C. At 23 0. the t o t a l count i s i n every case many times that found at 5°G, and i s conspicuously evident in the case of the samples obtained from the i n t e r i o r of the block of b u t t e r 0 Whilst the count obtained on these butters held at 23° G. i s as high i f not higher than the corresponding surface samples, the counts on the samples stored at 5°C» are consistently low i n the case of the Interior Samples» Grinding would appear to have exerted no influence on the bacterial development i n Interior butter. It w i l l be recalled that grinding was without influence on the b a c t e r i a l count of this butter when plated Immediately after processing. On the other hand, however, the grinding process i s seen to exert a marked eff e c t on b a c t e r i a l m ultiplication i n butter taken from the surface and held at 23°C., a difference recorded i n the case of these samples when plated immediately after sampling e The influence of the grind-ing process and temperatures of storage on the growth of yeasts and moulds i s c l e a r l y shown i n Table 2, No s p e c i f i c relationship betv/een b a c t e r i a l count and the incidence of surface t a i n t as dependent upon the grinding process, i s found to exist, Table 2 0'Although the butter was of high quality, i t i s to be seen that surface taint developed i n seven of the experimental samples. The majority of the re-maining samples retained their o r i g i n a l q u a l i t y . Certain of th« samples evidenced other defects which are recorded i n Table 2 6 I t i s to be es p e c i a l l y noted that surface t a i n t occurred only so i n the unpacked samples, and developed after storage at both 5"c, and 23 C. Other defects were confined p r a c t i c a l l y to con-tainers h a l f - f i l l e d with packed butter* In the case of the pa r t i c u l a r butter used i n the experiment, the development of surface taint appeared not to be conditioned by the grinding process, but to depend i n great measure on the procedure adopt-ed f o r the storage of the samples* I t would appear that aeration i s a determining factor for the occurrence of surface t a i n t * Apparently b a c t e r i a l growth of i t s e l f does not necessarily account f o r the development of the defect* The occurrence of surface t a i n t appears to depend on a number of closely.integrated b i o l o g i c a l , chemical, and physical factors* From these experiments i t would appear that "aeration" acts as a trigger, setting i n motion the chain of events which ultimately results in.the development of surface t a i n t , providdd the stage for i t s development has been set. Experiments 2 and 5* The experiment reported upon above was repeated on two subsequent occasions with s l i g h t changes i n experimental procedure. In the l i g h t of the results obtained i n Experiment 1, the method adopted f o r the storage of samples was r e s t r i c t e d to unpacked butter i n h a l f - f i l l e d containers at 5° and 23° C. In addition to the media employed i n Experiment 1, a l l samples were plated on Purple Lactose Agar i n an attempt to differentate further the types of microorganisms present under the d i f f e r e n t experimental conditions• In Experiment 2, the butter employed was obtained from a churning of butter which, although placed i n F i r s t Grade on m 63. ». the wholesale market, developed surface t a i n t when held i n prints under r e t a i l conditions. The butter for experimental purposes was taken from a 56 pound box of this churning held i n whole-sale cold storage© The butter employed i n Experiment 3 was also of F i r s t Grade; no report of surface t a i n t development i n prints on the r e t a i l market was reported© Discussion, The results recorded i n Table 3 show c l e a r l y that from a b a c t e r i o l o g i c a l point of view these experimental butters were of lower q u a l i t y than the butter employed i n Experiment 1, The butter employed i n Experiment 2 was much higher i n t o t a l count than that employed i n Experiment 3, Butter 3 as judged by Yeast and Mould Count would be considered the least desirable of the three samples from the view point of keeping quality, although i t s b a c t e r i a l content was not as high as that of Butter 2© The influence of grinding on butter as recorded by the counts obtained immediately after sampling d i f f e r from that reported i n the case of Experiment 1, In the second experiment grinding results i n a decreased t o t a l count p a r t i c u l a r l y on the samples from the Interi o r of the block. The count obtained on Purple Lactose Agar p a r a l l e l those obtained on Standard Agar, although at a lower f i g u r e , In this experiment, grinding appears to be without influence on b a c t e r i a l count, using either type of medium. In this instance the counts obtained on the respect* ive media are p r a c t i c a l l y i d e n t i c a l . The findings recorded after incubation for a period of seven days at 5 " and 23°C, respectively are d i f f i c u l t to i n t e r -pret • Although Butter 2 was considered higher i n i n i t i a l t o t a l - 62 -b a c t e r i a l counts than Butter 1, the samples taken after stor-age at both 5° and 23"G showed marked diminution i n count, a finding at variance with that obtained i n Experiment 1• Considerable b a c t e r i a l growth i s to be seen i n the • case of Butter 3, Table 3, p a r t i c u l a r l y i n the samples taken from the Interior of the butter and held at 5 C, The data ob-tained for the changes i n b a c t e r i a l types on storage as reveal-ed by plating on Purple Lactose Agar are of considerable i n t e r -est* In Experiment 2, i t Is to be noted that no a l k a l i producing colonies were indicated by this medium i n the case of the butters examined immediately after sampling* After storage, a l k a l i producing organisms are seen to constitute a considerable proportion of the b a c t e r i a l types appearing i n this medium, p a r t i c u l a r l y i n the sample held at 23 C. Their absence from the i n i t i a l plating of the butter may be due to t h e i r presence i n i n s u f f i c i e n t numbers i n the butter at that time, to be detected i n the dil u t i o n s employed« The p o s s i b i l -i t y that some condition of the butter prevented i n i t i a t i o n of t h e i r growth when plated cannot be excluded from consideration as a factor responsible for t h e i r apparent absence. I t w i l l be recalled that i n the case of Experiment 1, aeration was advanced as a possible explanation for the i n i t i a t i o n of growth and de-velopment of colonies• Although the aeration of grinding was apparently without influence i n Butter 2, the appearance of these types on Purple Lactose Agar after storage of the butter may well be explained by the aeration to which the butters are subjected during the holding period© Other factors of course —» f34 — may be at work. These organisms are pe c u l i a r l y fastidious with respect to i n i t i a t i o n of growth on a r t i f i c i a l media. Wo ade-quate explanation has so f a r been advanced f o r this phenomenon. Unlike the results obtained i n Butter 2, the i n i t i a l platings of Butter 3 on Purple Lactose Agar revealed a high pro-portion of a l k a l i producing types which are seen to constitute i n large measure the majority of the colonies developing on this medium from the samples stored for seven days at either 5 ° or 23°C. A finding of i n t e r e s t , yet d i f f i c u l t to explain, i s the occurrence of a high count made up c h i e f l y of acid-forming colonies i n the case of the Unground Butter taken from the i n -t e r i o r of the block and stored at 5°C. The growth of yeasts and moulds was quite pronounced i n the case of Butter 3 at both temperatures of storage• In the caisre of Butter 2, which showed a much lower i n i t i a l yeast and mould count, m u l t i p l i c a t i o n was only apparent i n the case of samples held at 2 5 ° C , and is p a r t i c u l a r l y to be seen i n the case of samples submitted to the Grinding Process• The incidence of surface t a i n t i s especially s i g n i f i c a n t , p a r t i c u l a r l y with respect to the question of the temperature to be employed for the storage of butter and with respect to the procedure to be adopted i n the storage of samples used to de-tect the occurrence of the defect. In' both Experiment 2 and 3, a l l butter samples held at 5°C. developed surface t a i n t . No defects were observed i n samples held at 2.3 G. This finding serves to confirm the conclusion drawn from Experiment 1 that "the occurrence of surface t a i n t appears to depend on a number of closely integrated b i o l o g i c a l , chemical and physical factors"• <» 65 « The f a c t that surface t a i n t developed i n a l l samples held at 5 0. confirms the finding reported i n Experiment 1, that, under the s p e c i f i c experimental conditions adopted i n this part of the work, the development of surface t a i n t appeared not to depend on the grinding process. One cannot conclude from these experiments that aeration due to grinding i s without influence i n the development of surface t a i n t i n the printing of butter using certain types of commercial equipment* Work done for a commercial firm experiencing d i f f i c u l t y i n the printing of t h e i r butter showed that this factor was of decis-ive importance under the conditions employed by the creamery i n question. In this instance 23°C. did not i n h i b i t the de-velopment of surface t a i n t * The f a i l u r e to demonstrate the c r i t i c a l influence of the grinding process on the development of surface t a i n t exper-imentally may have depended on an unfortunate or unwise selection of butter for experimental purposes. Any error i n this d i r e c t -ion was of course unintentional * It i s evident that i n these butters the stage had been previously set for the production of the defect* Apparently the only factor required was the setting of the t r i g g e r , the keeping of the butter at the temp-erature suitable for i t s development* The temperature employed by the Grading Service for the development and detection of flavour defects, and part-i c u l a r l y for surface t a i n t , i s approximately 23^0, The results reported i n this study show c l e a r l y that this temperature i s un-suitable f o r the development of surface t a i n t i n certain types of butter and that both 5*0 and 23°C. should be employed i n order to ensure the detection of surface t a i n t i n butter cap-- 66-able of exhibiting the defect* Experiment.4. The question of the direct contamination of butter with surface t a i n t producing bacteria as a s i g n i f i c a n t factor i n the development of surface taint i n butter printed from cer-t a i n types of equipment merits consideration, for i n this type of p r i n t e r , maintenance of proper sanitary conditions i s d i f f i -cult e In attempts to produce surface t a i n t experimentally by dir e c t inoculation of surface taint butter into a normal product Hammer and Derby (10) were regularly unsuccessful. Shutt (33), on the other hand, succeeded i n reproducing "svirface flavour" i n s t e r i l e butter inoculated with Pseudomonas fluorescens. Reference to the exact method of contamination used by him i s not given. In order to determine whether di r e c t contamination of high q u a l i t y butter with surface t a i n t producing microorganisms results i n the development of the defect, the following exper-iments involving the d i r e c t smearing of butter with an a c t i v e l y growing culture of the s p e c i f i c microorganism were carried out. The influence of changes i n the physical nature of butter — p a r t i c u l a r l y the influence of reworking — on the development of the defect under these conditions of contamination were also studied. For the purpose of t h i s experiment, several pounds of butter of exceptionally high q u a l i t y taken from a 56 pound box were obtained. Only butter from the i n t e r i o r of the block was employed. Using aseptic precautions, the block of butter was divided into three equal portions• One portion - Portion 1 -67 -served as the control. Portion 2 was inoculated with Proteus ichthyosmius (Campbell). Portion 3 was inoculated with a species of Pseudomonas (Campbell). Each portion was then div-ided into two parts, one of which was sampled d i r e c t l y and the other put through a s t e r i l e meat grinder, simulating the con-ditions encountered at times i n the commercial printing of butter, p r i o r to sampling. T r i p l i c a t e samples of each of the resulting six parts were then taken, one of the three for immediate pl a t i n g , and the others for storage at 5 C. and 23 C. respectively, prior to plating.c:"; Standard Agar, Malt Agar, and Purple Lactose Agar were employed for the microbiological analysis of the samples. The results of the determinations are given i n Table 4. Discussion. The high q u a l i t y of the butter employed i n this ex-periment as based on Grading Standards i s confirmed by the results of microbiological analysis as shown i n Table 4. The ef f e c t of grinding on the b a c t e r i a l count of the Control Butter i s sim i l a r to that obtained i n the case of Experiment 1• The increased b a c t e r i a l count obtained i n the case of the Control Butter when subjected to grinding confirms the results reported i n Experiment 1. Whilst a similar effect i s to be seen i n the case of the portion inoculated with Prot« ichthyosmius, the increased count due simply to the grinding process i s not to be seen i n the case of the butter contam-inated with Pseudomonas putrefaciens• This finding i s similar to that recorded i n the case of the butters used i n Experiments J c 0 . a 0 > •K C-t-0. j (0 •a (U a • -S5-C a IX a H *4_ 13,00C 3 o e» CM H <i o o CD. CO CO s 150,000 288,000 J c 0 . a 0 > •K C-t-0. j (0 •a (U a • -S5-C a IX 9 J Q . - S ^ 3 o o • 3 O PH o • o to ft H O O to ft <*• rH o o o ft o o GO ft CO CO o O o ft oo to o o o «l to Oi o o o ft to co CO J c 0 . a 0 > •K C-t-0. j (0 •a (U a • -S5-C a IX 3 J 1 i < . C 4= P o » • -p 9 Pi O O o ft CD O O O ft rH H O O <tf ft to o o o ft o o o «5 o o o ft o o CO o o o ft o o to J c 0 . a 0 > •K C-t-0. j (0 •a (U a • -S5-C a IX 3 J 1 i < . C 4= P o CD o EH o o o to O O O ft o to O o <* ! ft to t-O O O •t o to r-l ft •• to ... a • o o to to CO ft o o o ft . < o to • to J c 0 . a 0 > •K C-t-0. j (0 •a (U a • -S5-C a IX 1— as H CCi fl cb -=8 o o o o O J c 0 . a 0 > •K C-t-0. j (0 •a (U a • -S5-C a IX o CD u CD . ft 1 1 I •H " O • ' a ca. P4 . .9 -CQ -d O i cd _£S 9-CD rH o % ' ' ' « o o , H <! to co *> H to 03 to HI o o o ft . • •'<_' O o o ft .m ca o o °» 02 CO to ft HJ. o o to «t £> H to ' ' ' « o o , O PH r: •'• , • , lO 03 to ft H o to c. ft Oi H o o • o ft o o o> ft to to o o o ft o c-to 03 O o © 03 to to t» r. o o to H to ,ned after 7 days at <s 8 . •p O ; :& - - o O o « 03 © "I O ... tO. :. o o o ft o o to PS ° •' -if H 31. -P 0 o 01 EH © o — o © IO ft (X) 03 . o o o •*> o o O O ©"• •* o o • : © ' ft 02 . o" . •• o o ft to to to a, H o o o ft o o to +5 rd a p. fi> o © o ' o © • © • © +5 O Q> <k CD •t i ' ! • i CD +. •ri o PIES •H « iH CO <B fi ' CD +» Pi EH •H « Vt CQ CD ft -d ® • •HI a <H fcri • •rt « •d CQ o . ,-d-- . CD e •H EH -<!H « •H ca >d. o :, a'. a .y n <j © o 1 o to * to O O O ft <* 0> O o to ft o o O ft o co to o o o ft to _-g PJ « O a O PH. o IO o o lO ft to to o o o o o to ft 9 o o o o oo to o o o to e-6-Examined Immediately 6 <! « CQ o -p o o a •P O Pt PH o o o o ft CO o o o ft o to o © o *> o o o o ft © to 03 O o o ft © oo H 6 <! « CQ o -p o o H £0 o EH to CO o o e-•> CO to o o o a to to o o o ft to o o o • a o o to O O O ft o to •p -d aj <s CD O o o o o CO ft 05 © CQ © o o . a « r. O Pi Pi ffl -P -P Pi •P O a o — I-i . & ° Pi CD p, 0) >d +5 -P Pi Pi -P -rl O fl u o i CP CSv_, tn 'd • d CD+=>S -p o e Cd fH TS1 — U r-i f4 O • CD !_ ao += O _3 -P — "£ 9 *^ p >d '• «_> +» p to" Pi OHfM Pj ' a) -d fl S 0 •p c; o^-S"-4s -H Q-P-S! fl Pt Pi-rl9 -d rrf 5 | += O +5 ffl fl Pi-H J PQ-H &© CD ~S +5 . u _Jd in CD'd fl "S ~-+> p! O _2 -P H- H O S r« fl Pi Pi TH fl PQ t_ & & — 69 — 2 and 3, which f a i l e d to show an Increased count on grinding© It may well be that the s p e c i f i c type of microorganism pre-dominating i n a butter may determine whether or not an increased count i n butter results from the physical changes induced by grinding,, The apparent absence of alkali-forming microorganisms on plates made from the Control Unground Butter, and their pre-dominance on plates made from the same butter subjected to grinding i s a finding of considerable importance* Whatever the explanation, grinding results i n the appearance on plates of microorganisms which had previously f a i l e d to develop. The suggestion made i n explanation of the findings recorded on Experiment 1, that aeration may be an important factor i n this connection, i s thus rendered more plausible• Neither of the Control Butters held at 5° or 23°C« shov/ed a marked increase i n b a c t e r i a l count when compared with the results obtained i n the case of the infected butters• Microbial growth i n the samples inoculated with Proteus ichthy-osmius was indeed marked© Both Control Butters maintained their o r i g i n a l quality i n so f a r as flavour i s concerned, when stored at either 5° or 23°C. A l l the contaminated samples developed defects• Surface t a i n t was found i n a l l the samples held at 5°C. and occurred i n the case of the butter inoculated with Proteus ichthyosmius and submitted to the grinding process at 25"G. The other sam-ples held at this temperature were graded rancid or unclean* Rancidity often develops as an aftermath of surface t a i n t * The results of this experiment show c l e a r l y that - 70 -direc t contamination of "butter by smearing with surface taint producing bacteria results i n the development of the defect 0 M u l t i p l i c a t i o n of these organisms i n butter has also been dem-onstrated. The i n a d v i s a b i l i t y of storing butter at 5°C. as a preventive of surface t a i n t development i s again confirmed. - 71 -FART II C,._ - Experimental Churnlngs(1941) Experimental. Because of the d i f f i c u l t y encountered i n Part II A i n successfully and consistently producing surface t a i n t exper-imentally, attempts were made to discover the conditions unwit-t i n g l y set up i n previous work, which had exerted an inh i b i t o r y e f f e c t on the development of the defect. Experiments designed to determine the influence of the nature of the medium employed . for growth of the organisms used to inoculate the butter, the source of the cream supply, and the method of Infection were undertaken with t h i s object i n view. The influence of a c i d i t y and neutralIzation on the development of surface t a i n t was re-• investigated. In t h i s series of experimental churnings the procedure adopted i n the work reported under Part I I A was i n large meas-ure followed. Samples, however, were taken at three stages of working only, as follows:-I. S l i g h t l y worked. I I . Underworked. I l l * Thoroughly worked* Bacteriological examinations of the butter samples were not carried out. The microorganisms used i n t h i s part of the work were Ps. putrefaciens (Hammer), Prot. ichthyosmius (Hammer), Prot. ichthyosmius (Campbell), a species of Pseudomonas (Campbell), and two strains of Ps• putrefaciens recently isolated from surface t a i n t butter produced i n B r i t i s h Columbia. - 72 -Influence of Medium for Growth of Inoculum on Development of Surface Taint» In the l i g h t of work done on the influence of the nature of the medium - p a r t i c u l a r l y with respect to nitrogen and carbon supply - on the metabolism of the surface t a i n t bacteria, and reported upon i n Part III of this thesis, invest-igation of the effect of the nature of the medium used for the growth of the organism employed i n experiments on surface t a i n t production i n experimental churnings was undertaken. The media employed were:- • Tryptic Casein (Commercial) Digest Broth (Cole) (7) T.CeD B. Davis Broth.(Davis) (9) Tryptic Casein (purified) Digest Broth Tryptic Casein (Purified) Digest Broth.+ 0.5$ Glucose. Mode of Infection.. • In conjunction with the investigations on the i n f l u -ence of the type of medium on surface t a i n t development, exper-imental churnlngs were carried out i n order to determine whether or not the mode of i n f e c t i o n of the butter with the surface taint organism was an important factor governing the develop-ment of the defect. Prior to pasteurization, the cream was divided into two equal portions. The usual method of inoculation via the wash water'as practised under Part II A was employed i n the case of one portion. Contamination of the second portion was e f f e c t -ed by inoculation of the cream one h a l f hour pr i o r to churning• Discussion. As may be seen i n the results of Experiments 1 - 7 , . - 73 - • . . surface t a i n t was produced i n the majority of cases irrespect-ive of the' medium employed for growth of the inoculum, Tables 5 - 8, It appears that both Davis and Tryptic Broths favour the development of surface t a i n t i n butter. Therefore i t i s to. be concluded that the nature of the nitrogen source as pres-ent i n the two different media, has no influence on the product-ion of the defect. The results of Experiments 6 and 7 show c l e a r l y that the presence of glucose i n the medium used for growth of the organism does not i n h i b i t the development of surface taint i n butter. The method of inoculation as studied i n Experiments 1, 2, and 3 also appeared to have l i t t l e effect on the production of the t a i n t . Except i n Experiment 1, there was no marked d i f f -erence i n the development of surface t a i n t when either of the two methods of i n f e c t i o n were employed. In Experiment 1, i n -oculation of the cream appeared to favour the development of surface t a i n t . In this series of experiments, the degree of working had l i t t l e influence on the development of surfacd t a i n t , Tables 5 - 8. In Experiment 4, using Prot. ichthyosmius (Campbell) the butters held at 5 C. for a week, and then raised to 23 C , prior to grading, gave a very strong odour of ammonia when the sample jars were f i r s t opened. Fif t e e n minutes l a t e r , however, they were regarded as characteristic surface t a i n t butters. This phenomenon may be explained by the supposition that the surface t a i n t was masked by the strong ammoniacal odour but was l a t e r revealed, a f t e r the more v o l a t i l e ammonia had escaped^ 74 following the opening of the samples. However, the influence of aeration on the development of the defect, Is not to be over-looked. It may well be that the admission of oxygen even at thi s stage, acts as a trigger, setting off the chain of events leading to the development of the defect* The importance of aeration at a l l stages i n the manufacture and storage of butter i t s r e l a t i o n to the development of defects is a problem requir-ing further investigation. The results recorded i n Tables 5 - 8 , offer conclusive evidence that microorganisms other than Ps. putrefaciens are capable of producing the cha r a c t e r i s t i c surface t a i n t defect i n butter when inoculated either v i a the cream or wash water© The fact that i n this series of churnings, no d i f f i -c ulty was encountered i n the development of surface t a i n t exper-imentally shows c l e a r l y that f a i l u r e to obtain surface taint consistently i n Part II A was not dependent upon the nature of the medium employed for the growth of the causative organism nor upon the mode of i n f e c t i o n . ' Source of Cream Supply. The cream used f o r Experiments 1 — 7, Cream Source 2, was of d i f f e r e n t o r i g i n than that employed i n Part II A, Cream Source 1. Whether or not the source of cream supply was a fac t -or influencing the incidence of surface t a i n t i n the experiment-a l cburnings was determined by malting butter under i d e n t i c a l conditions, employing cream from both sources, Experiments 8 and 9, Table 9. Tryptic Casein (Commercial) Digest Broth was used as the medium for growth of the inoculated organism i n these and subsequent experimental churnings « - 75 -Results. Prom the results obtained i n Expermments 8 and Qf i t can r e a d i l y be seen that the source of cream supply had some influence, although s l i g h t , on the s p e c i f i c nature of the putrid defect developing i n butter* With Ps. putrefaciens, there was no apparent ef f e c t , but with Prot• ichthyosmius (Campbell) the characteristic surface t a i n t defect occurred only under certain conditions of working and holding i n butter . made from Cream 1, whereas In the case of butter made from Cream 2, the defect occurred under a l l experimental conditions These findings suggest that the difference i n the cream supply may have been responsible i n a measure fo r the f a i l u r e to produce the chara c t e r i s t i c defect i n butters made under Part I A. Ac i d i t y and Neutralization. In order to reinvestigate the s p e c i f i c influence of a c i d i t y and neutralization on the production of surface t a i n t , the following experiments were carried out. Ac i d i t y . Raw sweet cream was divided into three equal parts, pasteurized and cooled. S t e r i l e l a c t i c acid i n s u f f i c i e n t quantity to increase the o r i g i n a l t i t r a t a b l e a c i d i t y by 0 e l $ and 0.2$, respectively, were added to two of the f r a c t i o n s . After being held overnight i n the icebox, the creams were churned employing the procedure previously described — Exper-iments 1.0 and 11, Tables 10 and 11. Neutralization. In this series of churnings the procedure followed was s i m i l a r to that employed i n the "Acidity Series", except that the l a c t i c acid was added prior to pasteurization, and the varying degrees of neutralization employed were obtained by the addition of Wyandotte CAS, added at a temperature of 90°P., Experiments 12 and 13, Tables 12 and 13. Further studies on the influence of a c i d i t y and neu-t r a l i z a t i o n were undertaken i n Experiments 14 and 15, Tables 14 and 15• Raw sweet cream was divided into three portions• One portion served as the control — Cream 1• The other two portions were a c i d i f i e d to 0.32$, a c i d i t y , following i n one case the procedure outlined under the "Acidity Series" Cream 2, and i n the other, the procedure used i n the "Neutralization Series" — Cream 3. Cream 3 was neutralized employing Wyandotte CAS. Pis cuss ion. Although a c i d i t y has been claimed to exert an inhib-i t o r y influence on the development of surface t a i n t , the results of Experiments 10 and 11 show c l e a r l y that experimental butters made from cream at an a c i d i t y as high as 0.3^ regularly develop-ed the c h a r a c t e r i s t i c defect when the water used for washing the butter was infected with certain surface t a i n t producing bacter-i a • Contaminat1on with Ps• putrefaciens, Prot. ichthyosmius (Campbell) regularly resulted i n i t s development. At t h i s acid-i t y , Prot. ichthyosmius (Hammer), however, f a i l e d to produce the defect. Neutralization of the L a c t i c Acid of the cream did not appear to have any influence, neither i n h i b i t i n g nor stimulating the. development of the defect when Prot• ichthyosmius (Campbell) « 77 ~ Ps. putrefaciens (Hammer), and Pseudomonas (Campbell) were used as the contaminating organisms, Tables 12 and 13» In the case of Prot 0 "ichthyosmius (Hammer), neutral-i z a t i o n of the cream did not lead to the development of surface t a i n t , but instead resulted i n the development of other equally undesirable defects. Over neutralization of the cream resulted i n a neutralizer odour i n the case of Prot. ichthyosmius (Campbell)• With Ps. Putrefaciens, however, the characteristic surface t a i n t odour was produced® From this series of experiments i t i s d i f f i c u l t to draw s p e c i f i c conclusions as to the influence of holding temper-ature on the development of surface t a i n t . 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L i t t l e d e f inite information as to the nature of the stibstance produced i n butter by ba c t e r i a l action and responsible for the undesirable odour characteristic of Surface t a i n t , i s as yet available*, Derby and Hammer (10) state that "Protein de-composition of a f a i r l y d e f i n i t e character i s involved i n the development of surface t a i n t . " , but make no reference as to the sp e c i f i c nature of the degradation. Dunkley (11), i n work on the chemistry of surface t a i n t , shows that the "sweaty feet" odour produced i n milk by Ps. putrefaciens i s v o l a t i l e with steam from an acidic solution, suggesting that the compound pro-ducing t h i s odour i s of an acidic nature• He claims that i t i s a non-nitrogenous compound* Prom time to time, as the work reported upon pro-gressed, several hypotheses postulating the possible nature of the odoriferous substances and the mechanism of t h e i r formation by the b a c t e r i a l breakdqwn of the protein material i n butter were advanced. Experiments designed to substantiate or d i s -prove the v a l i d i t y of these hypotheses were performed. Indole Hypotheses. H i s t o r i c a l * In work carried out by Campbell (3) on the hypothes-i s that the odour characteristic of surface taint was due to the presence of amines produced from amino acids by the causative microorganisms, melted butter to which indole and ethylamine hydrochloride had been added was considered by competent grad-ers to be strongly suggestive of the odour of surface t a i n t * In further work employing d i f f e r e n t combinations of possible - 91 = odoriferous substances, butters containing indole as one of the added compounds always swere judged as closely resembling sur-face tainto• In compilation of work on the production of indole by bacteria, Stephenson (36) states that i t s formation i s i n h i b i t -ed by the presence of carbohydrate, glycerol, and calcium l a c -tate « Campbell (4) found that butter made from neutralized cream i n which acid had been formed by the Lactic Acid Bacteria f a i l e d to develop surface t a i n t , a finding which he interpreted as substantiation.of his "Indole Theory" for Surface Taint pro-duction i n butter• He attributed the i n h i b i t i o n of Surface Taint development as due to the lack of Indole formation which he claimed was due to the presence i n the cream of the carbo-hydrate , lactose, and the calcium l a c t a t e , formed on n e u t r a l i -zation, acting as inh i b i t o r s i n the manner described by Stephenson. Woods (38) reported that the degree of aeration i n f l u -enced the rate and completeness of the reaction of the quant-i t a t i v e production of indole from t^ptophan by thick washed suspensions of Bact«, c o l i . • Aeration was found to increase indole formation. Pildes (14) i n studies on the production of indole from t'yptophan by Bact. c o l i using the washed c e l l technique, found that the stispensions tahen grown i n the presence of typtophan were about twenty-five times more active than when grown i n the absence of tryptophan. Evans, Handley, and Happold (13), i n working on the production of "tryptophanase" (the coupled oxidative deamin-ation and decarboxylation i n an enzymic system which i s - 92 -s p e c i f i c for the iTyptophan- inc lo le raction) by Esch.coli found that the presence of tryptophan i n a medium i s essential for the production of the tryptophanase system,, It was found also that t h e presence of glucose i n the growth medium has an i n -h i b i t o r y e f f e c t on the formation of the tryptophanase system, thus preventing t h e production of indole by the c e l l suspensions However, once the tryptophanase system has been set up by growth i n a sugar free tryptophan medium, glucose has no i n h i b i t o r y e f f e c t on the production of indole by the c e l l suspensions Experimental. Prior to the setting up of experiments designed to prove or disprove- the Indole Theory for the development of Sur-face Taint i n butter, a study of the s e n s i t i v i t y of various tests employed f o r the detection of indole and the determination of the indole-producing a b i l i t i e s of the microorganism under ideal conditions for i t s elaboration i n b a c t e r i o l o g i c a l media were undertaken. The s e n s i t i v i t y of Nencki's reagent, the Weyl-Legal reagent, Ehrlich's para-dimethyl-amino-benzaldehyde te s t , the procedure of Woods (38) and the xylene extraction method of Happold and Hoyle (19) for the detection of indole was deter-mined. The method of Happold and Hoyle gave by far the most sensitive and delicate reaction. Employing t h i s test, the indole producing a b i l i t i e s of the microorganisms used i n this study were determined after growth f o r varying periods of time i n Tryptic Casein Digest Broth - a medium found suitable for indole production. Tests were performed on cultures inoculated at 23°, 30°, 31° C , res-- 9 3 -pectively. The results are shown i n Table 17. Proteus ichthyosmius (Hammer) i s seen to give a d i s t i n c t l y positive test at a l l temperatures, 30° and 37°C, however. appeared more suitable for indole development than 23° G. Ps* putrefaciens and Proteus ichthyosmius (Campbell) both f a i l e d to give positive t e s t s . Table 17. Studies on the Inhibitory effect of glucose and Ca-lactate on Indole production were of necessity limited to Proteus ichthyosmius, the only culture giving a positive Indole Test i n i d e a l a r t i f i c i a l media• In order to determine the ef f e c t of glucose on the pro-duction of indole, experiments were set up whereby Prot 8 i c h -thyosmius was inoculated into both T.C.D.B. and Tryptone Broth containing increasing percentages of glucose. Tryptone Broth, a nitrogen source commonly employed for the Indole Test, served as a control f o r the T.C.D.B. The ef f e c t of the presence of glucose i n the medium used for the growth of the culture prior to inoculation into the test broths, on Indole production, was determined by growing the organism i n T.C.D.B., containing vary-ing percentages of glucose. The results of these determinations are given i n Table 18. Indole production was observed i n T.C»D.B• containing 0*3$ glucose, but not with 0.57$ glucose. In tryptone broth, however, no culture containing more than 0.1 $ glucose gave a d e f i n i t e l y positive t e s t . Glucose i s thus shown to exert an i n h i b i t o r y influence on indole formation from Tryptophan by Prot. ichthyosmius, confirming the findings of Stephenson on B. c o l i . The presence of glucose i n the medium from which the culture i s inoculated f o r the detection of indole is seen to be - 94 -without influence on i t s production* The e f f e c t of Ca lactate on indole production was de-termined by inoculating Prot. Ichthyosmius into iyyptone broth containing varying amounts of Ca l a c t a t e . I t was found that Ca lactate p a r t i a l l y inhibited the production of indole at 0.5$ concentration, a positive t e s t , however, being obtained i n the presence of as high as 2.0$ Ca l a c t a t e . Taken as a whole, the studies on I ndole production lead by inference to the conclusions that indole i s not an essential constituent of the characteristic Surface Taint odour. Although only one of the surface taint producing bacteria could be i n -duced to elaborate indole on a r t i f i c i a l media, the p o s s i b i l i t y that indole formation plays a part i n the development of surface t a i n t i n butter cannot be e n t i r e l y dismissed from consideration. The i n h i b i t i n g effect of both carbohydrate and Ca lactate de-monstrated i n this experiment not only confirms work done by Stephenson on other microorganisms, but makes impossible the complete dismissal of the Indole Hypothesis as an explanation of surface t a i n t development i n butter. If indole formation i s a requisite of surface t a i n t development, i t s presence should be capable of detection i n butter showing the defect. Employing the sensitive reaction of Happold and Hoyle, the sera obtained from those butters described i n Part I I C , Experiments 1, 2, 3, & 6, Tables 5, 6, & 8, and exhibiting the defect were tested for the presence of indole. Further, i n the case of Experiments 3 and 6, sera from aliquots of the butters taken at the time of manufacture, prior to the development of the defect, were taken, placed i n 95 -> Table 17. -Drganism Medium Temperature Time Reaction, Prot. ichthyosmius 23 G • 24 Hrs® + 23 C 9 48 Hrs • + + 30° C. 24 Hrs • + +-30° 0. 48 Hrs . + +• + 37°C. 23 Hrs« + i-37°C. 48 Hrs e Ps . putrefaciens (Hammer) 23° C. T • C • 3D o 3 • 24 Hrs • S3 C« 48 Hrs.© 30°C. 24 Hrs • -30 °C. 48 Hrs «.. 37°C. 24 Hrs © 37 °C. 48 Hrs • Prot• ichthyosmius 23°C. b (Campbell) 24 Hrs ® 23°G. 48 Hrs « -30 °C. 24 Hrs « «© 30°C. 48 Hrs o tm 37°C. 24 Hrs o eo 37 PC . 48 Hrs . K3 - 96 -Table 18. 3r gani sm Medium Time 1 Reaction Prot. ichthyosmius Tryptone B.+0.1$ glucose 52 Hps. (Hammer) 11 +0.2$ M 52 •* (+)? " +0.3$ 5 52 " " +0.4$ " 52 " CO " +0.5$ » 52 " CSS Tryptone Broth (Control) +• +• Prot. ichthyosmius (Hammer) T.C.D.B.+ 0.3$ glucose 72 Hrs. +-" " " " +-0.57$ " 72 Hrs. » " " " '» +0.85$ " 72 Hrs. T.C.D.B. (Control) + + + Prot. ichthyosmius (Hammer) T .C.D.B.+ 0o3$ glucose 52 Hrs. + + + it » H » x 0 # 5 7 ^ » 52 " + + + " » " » +0.85$ " 52 " + + + Prot. ichthyosmius (Hammer) Tryptone B .+ 0.5$ Ca lactate 52 Hrs • + + " B.+1.0$ 11 " 52 " 4 " B0+l4,5$ " " 52 »• + 11 B.+ 2. $ " » 52 "• + Tryptone B. (Control) + + + - 97 •-s t e r i l e tubes and incubated at 5°, 23°, and 30°C. for 7 Days and then tested f o r indole© In none of these sera was the presence of indole even suspected, thus rendering the Indole Hypothesis pr t i c a l l y untenable* Decarboxylation Hypothesis© Because experimental evidence did not substantiate the hypothesis that indole was an essential constituent of the characteristic surface t a i n t odour» consideration was given to the p o s s i b i l i t y that, a s p e c i f i c amine or combination of amines?. produced by decarboxylation of the amino acids by the causative microorganism, was responsible for the development of the defect*, Historical© Campbell (3) i n studies on the hypothesis that indole formation was the necessary reaction for development of surface t a i n t , reported that d i f f e r e n t amines i n combination with indole, approximated the defect * Gale (15), i n recent work on decarboxylation using the washed c e l l technique, and thus demonstrating the ensymic nature of the reaction, was able to form agmatine,,cadaverine, histamine, and . amine butyric acid from their respective amino acids using suspensions of Bact. c o l i . He found that the decarboxylases f o r the various amino acids varied i n t h e i r optimum pH a c t i v i t y values© The lower the pH of the medium within l i m i t s the high-er the a c t i v i t y of the washed suspension. He found also, that the enzymes are not produced i f the organisms are grown i n an amino acid free medium* Gale (16) also found that not only gram negative but also gram positive organisms are capable of decarboxylations - 98 -He showed that a s t r a i n of Sc. f e c a l i s had the a b i l i t y to de~ carboxylate only one amino acid, forming tyramine from tyrosine* However, i t was also capable of producing the amino acid o r n i -thine from arginine. Further he showed that putrescine results from the associative action of Sc. f e c a l i s and B. c o l l on arg-inine. The action of these organisms on arginine results i n ; (1) Decarboxylation of arginine to agmatine by Bact. c o l i (2) Breakdown of arginine to ornithine by Sc. f e c a l i s . (3) Decarboxylation of ornithine to putrescine by Bact 0 . coli© Experimental» The fa c t that the evidence obtained by G ale had shown that an acid reaction was essential for decarboxylation with resultant amine formation, lent support to this hypothesis. The demonstration of the associated action of microorganisms i n the formation of certain of the amines tended to substantiate th i s concept, especially when the conditions favourable to the production of surface taint i n butter are considered. Employing the technique described by Gale, decarboxy-l a t i o n experiments were performed, employing both Ps• putrefac-iens" and Prot • ichthyosmius on the amino acids arginine and h i s t i d i n e . In no case was any agmatine or hi s t i d i n e i s o l a t e d . Unless the conditions requisite for amine production from amino acids by species of the Genera Pseudomonas and Proteus d i f f e r from those essential for amine production by strains of B. c o l i , i t may be concluded that these organisms do not elaborate a decarboxylation enzyme system f o r these amino acids. The re-l i a b i l i t y of these findings i n the l i g h t of the d i f f i c u l t i e s - 99 -accompanying this procedure was confirmed when agmatine was readily isolated from arginine employing B. c o l i communior• Although decarboxylation of the amino acids h i s t i d i n e and ar-ginine , readily observed i n the case of members of the coliform gnoup and for this reason selected for study of the decarboxylase a c t i v i t y of the gram negative surface t a i n t producing bacteria, did not occur, the p o s s i b i l i t y remains that these organisms may be capable of decarboxylating other amino acids® Deamination Hypothesis . Owing to the f a i l u r e to obtain experimental evidence substantiating either the Indole or Decarboxylation Hypotheses for the production of surface t a i n t , and hypothesis postulating the formation of hydroxy and unsaturated acids from amino acids by deamination was considered as a possible explanation of the nature of the mechanism determining the elaboration of the sur-face t a i n t odour. The evidence put f o r t h by Dunkley (11) that the odoriferous substance i s acidic i n nature lends some support to this hypothesis« The work of Sasaki (32) on the conversion of tyrosine into parahydroxy phenyl l a c t i c acid by strains of Prot. vulgaris, and that of Raistrick (30) on the formation of TJpocanic acid from h i s t i d i n e by species of the coliform group of bacteria strongly suggests that the gram negative micro-organisms associated with surface t a i n t development may be cap-able of deaminating amino acids, and that one of these acids, formed on deamination, or combinations thereof, may be res-ponsible for the c h a r a c t e r i s t i c surface t a i n t odour i n butter© Preliminary experiments designed to determine the v a l -i d i t y of t h i s hypothesis has shown that the undertaking of a thorough investigation of t h i s aspect of the problem merits consideration. - 100 -Summa r y and C on c l u s i on "s . The l i t e r a t u r e on the putrefactive deterioration of butter has been reviewed* Detailed descriptions of the microorganisms employed throughout the study are given* A new medium for the i s o l a t i o n of surface t a i n t pro-ducing bacteria i s described* Experiments on the detection of these organisms by t h e i r action i n milk are outlined* A technique for experimental butter making on a labor-atory scale was developed. In (Setermining the influence of the degree of working i n experimental butter making, i t was observed that surface taint or closely related odours and flavours occurred more often i n underworked butter than i n well worked butter. There was usually a decrease i n potency of the odour with an increase i n the degree of working. It was also found that there was a tendency towards a def i n i t e decrease i n b a c t e r i a l count as the extent of working increased* It was found that the associative action of surface taint and acid producing microorganisms resulted i n a higher incidence of undesirable odours and flavours than when the surface t a i n t producing organism alone was employed. Defects closely akin to surface taint were.encountered more oft en i n butter of low than i n butter of high s a l t content• In experiments on the influence of grinding on commer-c i a l butters i t was found that the methods used i n the sampling and handling of butter for t o t a l b a c t e r i a l count determinations - 101 -may be of paramount importance* Of equal i f not greater s i g -nificance was the finding that the yeast and mould count was influenced greatly by the method of handlings The results obtained on different butters suggest that an increase i n microbial population resulting from physical changes induced i n butter by grinding may be determined by the s p e c i f i c types of microorganisms predominating i n the butter* No s p e c i f i c relationship between b a c t e r i a l count and the incidence of surface t a i n t as dependent upon the grinding pro-cess was found to exist* Direct contamination of a normal commercial butter re-sulted i n the development of surface t a i n t * • The nature of the nitrogen source of the medium employed for -growth of the inoctilum as studied i n this work was shown to have no influence on the production of surface taint« It was found that the presence of glucose i n the medium f a i l e d to i n -h i b i t development of the defect* The method of inoculation was found to be without i n -fluence on the production of the t a i n t . The source of the cream supply may influence to a s l i g h t extent, the development of surface t a i n t * 0*3$ t i t r a t a b l e a c i d i t y of the cream did not i n h i b i t the production of surface t a i n t i n the subsequent butters made therefrom employing a surface t a i n t producing bacterium as con-taminant c Neutralisation of the cream had neither an inh i b i t o r y nor stimulatory effect on the development of the defect* Organisms other than Ps e putrefaciens have been shown - 102 -capable of producing surface t a i n t i n butter. The significance of the temperature selected for the holding of butter prior to grading i s emphasized. Reasons for the i n a d v i s a b i l i t y of storing butter at 5°G. as a preventive for surface t a i n t development are presented,, The occurrence of surface taint has been shown to de-pend on a number of closely integrated b i o l o g i c a l , chemical and physical factors and the significance of aeration as a determin-ing factor for i t s occurrence has been demonstrated. Results of work carried out i n an attempt to substantiate the Indole Hypothesis showed that i t i s p r a c t i c a l l y untenable as an explanation of surface taint i n butter. Evidence i s presented suggesting that decarboxylation of amino acids with resultant amine formation is not responsible for the development of surface t a i n t . Preliminary experiments to determine the v a l i d i t y of the Deamination Hypothesis postulating the formation of hydroxy and • unsaturated acids from amino acids by deamination showed that the undertaking of a thorough investigation of t h i s aspect of the problem merits consideration. - 10S References 1. Bergey, D . J . 2. Brown, A. M. 3. • Campbell, J.J.R, 4. 5, 8. 10, 11 Campbell, J.J.R. Campbell, M.L. Claydon, T.J. and Hammer, B. W. C u l l i t y , M,, and G r i f f i n , D.G. Davis, J.G. Derby, H.A., and Hammer, B, I , Dunkley, W.L. and Thornton, H.R. 12. Eckles, C. H. Manual of Determinative Bacteriology 5th E d i t i o n . New South Wales butter quality Agr• Gaz. N.S. Wales, 39: 843. 1928. Cited by Derby and Hammer (10). A study of surface t a i n t i n butter. A p r i l , 1939. Personal Communication. C l a s s i f i c a t i o n of microorganisms assoc-iated with surface t a i n t i n butter, A p r i l , 1940. Bacteriology of Butter. VIIIo Relationship of Achromobacter putrefaciens to the putrid defect of butter. Iowa State College of Agriculture Res. Bui. 267, 1939. Rabbito or surface t a i n t i n butter. J. Dept. ofAgr., Western A u s t r a l i a . Ser. 2, 15,: 137-147. 1938. Cited by Claydon and Hammer (6). Notes on the preparation of media, etc. The National Institute for Research i n Dairying, University of Reading. Bacteriology of Butter. IV. B a c t e r i o l -ogical studies on surface t a i n t butter. Iowa Agr. Ex. St. Res. Bui. 145. 1931. 2nd Progress report on an investigation of surface t a i n t butter. A case of putrid butter. Iowa Agr. Ex. St. Bui. 59, 50, 1901. Cited by Derby and Hammer (10). 13. Evans, iW.C, and Handle y, V/. R. C., and Happold, F.C. The tryptophan-indole reaction. 4. Some observations on the production of tryptophanese by Esch. c o l i . Biochem J. 35, No. 1 & 2, 207, 1941. - 104 -14. F i l d e s , P. 15 « Gale, E.F. 16. Gale, E.F. The production of indole by suspensions of Bact. 3911. Biochem J. 32, No. 9, 1600. The production of amines by b a c t e r i a 0 1. The decarboxylation of amino acids by strains of Bact. c o l l . Biochem J. 34, No. 3, 1940• The produ ction of amines by bacteria. 3. The production of putreseine from l ( - ) arginine by Bact. c o l i i n sym-biosis with Strep, f e c a l i s » Biochem J. 34, No. 6, 853, 1940. 17. Gilruth, J.A. Bacteriological examinations for the dairy science. N.Z. Dept. of Agr. 7th E r t . , p 89.1899. Cited by Derby and Hammer (10)• 18. Hammer, B.W., and :. . Yale, M.W. Development of the Escherichia-Aerobactes group of bacteria i n butter. I . Dairy S c i . Vol. 15, 199. 1932. 19. Happold, F.C., and Hoyle, L. CLIX. The quantitative determination of indole i n bact e r i a l cultures. Biochem. J . 28, No. 4, 1171. 1934. 20. Happold,« F.C., and Hoyle, L. CCXXVII. The coli-tryptophan-indole reaction. 1o Enzyme preparations and the i r action on tryptophan and some indole deriva-tives . Biochem. J . 29, No. 8, 1918. 1935. 21. Pierre i d , Macy and Combs. Microbiology of cheese-like flavours i n unsalted butter. Minnesota Tech. Bui. 97,,1934e 22o Hood, E.G., and White, A.W, Surface t a i n t butter. Canada, Dept. of Agr. Pam. 91, New Series 23. Hussong, R.V., Long, H.F., and Hammer, B.W. C l a s s i f i c a t i o n of the organisms import-ant i n dairy products « I I . Pseudomonas f r a g i . Iowa State College of Agriculture Res. Bui. 225, 1937. - y10f5 -24.Long, H.F., and Hammer, B . W . 25«Long, H.F., and Hammer, B.W• 26 oLong, H.F., and Hammer, B.1Y: 27,.Long, and Hammer, -B.W. 28 .Orla-Jensen, S. 29.Orla-Jensen, S. 30.Raistrick, H. 31.Sadler, W i l f r i d , and Vollum, R.L. . 32.Sasaki, T. 33. Shutt, D.B. 34.Spitzer, and P a r f i t t Bacteriology of butter. Ef f e c t of moisture dispersion i n butter on growth of b a c t e r i a 0 Iowa Agro Ex. St. Res. Bui. 247, 1938. Examination of butter with the Burri Smear Culture Technique» Iowa State College J. S c i . 12, 441, 1938, Bacteriology of butter© VII. E f f e c t of reworking butter on growth of b a c t e r i a . Iowa Agr. Ex. St. Res. Bui. 236, 1939. C l a s s i f i c a t i o n of organisms important i n dairy products » I I I . Pseudomonas putrefaciens. Iowa Agr. Ex. St. Res. Bui. 285, 1941. Outlines of Dairy Bacteriology — Russel and Hastings. 7th E d i t i o n , 1910, 154. The Lactic Acid Bacteria (in English) The conversion of h i s t i d i n e into uro~ canic acid by bacteria of the C o l i -Typhosus group. Biochem. J . 11, 71, 1917. An Investigationinto the possible correlation of the grading of butter with the b a c t e r i a l content of the same. The influence of conditions of bacter-i a l cleavage of proteins on the cleavage products e J . Biochem. 32:527, 1917. Contaminated water as a source of sur-face flavour i n pasteurised creamery butter. Sc. Agr., IX: 316, 1929. A study of the proteolytic action of spe c i f i c organisms and groups of or-ganisms i n butter made from graded cream. J . Dairy Sc. 12:1. 1929. 106 -35© Sproule and Hamilton 36• Stephenson, Marjory 37s Stephenson, M. and Gale, E.P. 38. Woods, D.D. ' 39. Wolochow, 11,, and Thornton, H.R. 40. Wolochow, H., and Thornton, H.R„ Canadian Dairy & Ice Cream Journal X V I . No. 2, Feb. 1937. Bacterial Metabolism 2nd Edition, 1939. Bacterial Deamination Biochem J. 31, No. 8, 1316, 1937. LXXVI. Indole formation by Bacteriam c o l i 1. The breakdown of tryptophan by washed suspensions of Bacterium c o l i . Biochem. J . Vol. 29, Wo. 3, 640,.1935. 1st Progress report on an i n v e s t i g a t i o n of surface taint butter. 2nd Progress report on an investigation of surface t a i n t butter. - 1 0 7 -Acknowledgements I wish to express ray very sincere thanks and appreciation to Dr. B. A. Eagles for his generous help and advice in every phase of thi s work, and to Mrs. George Yolkoff for her valuable aid and suggestions. To Mr. H, A. Mason, Dominion Dairy Produce Grader for the Province of B r i t i s h Columbia, I especially desire to extend my gratitude, Without his kind co-operation in grading the experimental butters the work could not have been undertaken with the confidence that the experimental gradings would b© authoritative. I wish also to thank Miss Nora Neilson for her suggestions and help i n laboratory work. Grateful acknowledgement i s made to the University of B r i t i s h Columbia for a Research Grant which made the work possible. 

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