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UBC Theses and Dissertations

The relation of certain lactic acid streptococci to a "slight-cooked" flavour in cheese 1928

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U.B.C. LIBRARY CAT. HO. I S a - / W - T a R ACC. /ff £ THE RELATION OF CERTAIN LACTIC ACID STREPTOCOCCI TO A "SLIGHT-COOKED" FLAVOUR IN CHEESE. HUGH LEWIS AUBREY TARR. A Thesis submitted for the Degree of Master of Science in Agriculture in the Department of Dairying. THE UNIVERSITY OP BRITISH COLUMBIA A April,1928 J TABLE OP COITTMT5 Page Introduction .. 1 Preliminary Studies 3 Experimental Cheese-making 5 The Detailed Cultural Study 6 Media 7 Methods 8 Table 1 . 11 " 2 13 " 3 14a Classification of the Organisms Studied. 15 Summary ................................ 18 Acknowledgment 20 References 21 Appendix I 23 THE RELATION OF CERTAIN LACTIC ACID STREPTOCOCCI TO A "SLIGHT- COOKED" FLAVOUR IN CHEESE. INTRODUCTION. It has heart known for some years that the aromas and flavours occurring in dairy products frequently result from the action of micro- organisms. Numerous experiments have been conducted the results of which prove that the lactic acid producing bacteria are, in the main, responsible for the desirable aromas and flavours which the higher grades of dairy produce exhibit. Conn (1) found that a micro-organism, named by him "Bacillus No. 41"» did not cause milk to clot, and rendered it only slightly acid, but gave a most pleasant aroma and flavour. He later (1) (2) showed that this Bacillus greatly improved the quality of butter made from cream ripened by its aid. Orla-Jensen (3) classified the aroma bacteria first studied by Storch, and gave to them the name Streptococcus cremoris. Hammer and Bailey (4) showed that the volatile acid produced by good start- ers was greatly in excess of that produced by cultures of Streptococcus lacticus, and later Hammer (5) classified two strains, which produced high volatile acid in milk, as Streptococcus citrovorus and Streptococcus para- citrovorus. Hammer and Sherwood (6) concluded that the volatile acid fig- tire was of considerable importance in determining the flavour and aroma of good starters, but that it was by no means the only factor. Notwithstanding the above, examples are not lacking which show that certain strains of lactic acid bacteria are capable of rendering dairy products unpalatable. Orla-Jensen (3) states that Streptococcus lactis, "gives milk and cream either a purely acid taste or an unpleasant flavour"; and also (7) that certain strains of the lactic acid bacteria impart to milk a "tallowy" and frequently a "burnt" or "malty" taste. Sadler (8) noted that a strain of Bacterium lactis acidi (Leichmann), which appeared true to type according to morphological and cultural determin- ations, produced a "burnt" or "caramel" flavour in milk. Leitch (9) iso- lated two organisms of the Streptococcus lacticus type from different sources and found that they imparted a "burnt" flavour to the curd of cheese and to butter made from cream ripened by their aid. Hanmer and Cordes (10) studied "burnt" or "caramel" flavour in dairy products, and tentatively named the causative organism Streptococcus lacticus var malti- genufl. McDonnell (1899) cited by these investigators, showed that an organism of the lactic acid group, which he named Bacterium lactis acidi maltigenum, produced a malt like flavour in milk. Sadler (11) succeeded in isolating numerous strains of Streptococcus lactis (Lister) from a large consignment of butter which had proved to be unmarketable because it exhibited a "specific and undesirable flavour". A high percentage of these strains produced a caramel flavour and aroma in milk. Experimental butter and cheese made with the assistance of one of the isolated strains developed an identical flavour and aroma. Hucker and Marquardt (IE) used strains of Streptococcus paracltrovorus (Hammer) in conjunction with a commercial starter to manufacture Cheddar cheese, and found that the resxilting product developed a "pungent" or "sli^vtly bitter" flavour. Cheese made using the commercial starter alone developed no such abnormal flavour. Cheese made from pasteurized milk showed the abnormality more markedly than did cheese made from raw milk. Daring the late summer of 1927 certain Cheddar cheese made in the Dairy Department of the University of British Columbia were observed to develop a "slight-cooked" flavour. The flavour was first noticed in the cheese some weeks after the date of manufacture. As the cheese were made from pasteurized milk the question arose as to whether the flavour could be traced to the treatment of the milk. In the absence of definite evidence it can only be stated that no such trouble had been noticed before although the milk for the cheese-making had been similarly treated for four years. Another aspect appeared to merit attention. In the making of cheese from pasteurized milk the starter employed is of even greater importance than is the case when using raw milk in the usual way. Was the starter implicated? In an attempt to settle these possibilities the work with which this paper deals was begun. PRELIMINARY STUDIES. In the manufacturing process of the cheese under examination a composite starter consisting of fifty percent of each of D144 and S2 had been used. For convenience these starters will from now on be respect- ively designated as starter "A" and starter "B". Each starter was of itself a mixed culture. Both gave a clean acid clot in milk with an aroma and flavour typical of a good starter, and neither evidenced any abnormal characteristics. The mixture was being employed on account of the excel- lent results which had previously attended its use. Using appropriate media (Whey Agar) (14) and methods quantitative bacteriological examin- ations of each of the two starters were made and a large number of colon- ies isolated and inoculated directly into litmus milk. From starter "A" fifty-eight cultures which clotted milk were isolated; of these forty- eight formed chains identical with those given in milk by Streptococcus cremoris as shown by Orla-Jerasen's photo-micrographs (3). Seventy-one strains clotting milk were isolated from starter "B" and of these sixty-five proved to be streptococci with morphology like that produced by Streptococcus cremoris in milk. It was noticed that certain cultures isolated from these starters gave a "pungent" aroma in milk. Such strains when inocu- lated into flasks of sterile milk caused the milk to clot with an acrid flavour which left a "dryness" or "roughness" upon the palate. Four such strains were retained from each starter for mare detailed study and were recorded as followst- From starter "B":- B37, B39, B43, B3. " "A":- A10, A15, A39, A43. In view of the pungent aroma given in milk by some of these organisms the cheese having the "slight-cooked" flavour were examined by dropping, under aseptic conditions, small pieces of each into sterile milk. Although the cheese were from two to three months old at the time, the milk clotted rapidly at 23° C, with a flavour and aroma seemingly identical with the flavour and aroma produced by certain of the streptococci isolated from the two starters. Stained preparations made from the freshly clotted milk inoculated with the cheese direct showed in every case numerous strepto- cocci similar in form to Streptococcus cremoris (3). The obvious procedure at this stage was to isolate the prevail- ing organisms in the milk inoculated with the Cheddar cheese direct. In explanation of the fact that this was not done I can only suggest that my interest had become concentrated on the specific aroma and flavour pro- ducing strains which had been recovered from the starters. Most unfortun- ately while concentrating upon these organisms I permitted myself to throw out the critical milk cultures which had been obtained by the mass cheese inoculations. Too late I discovered the inexcusable blunder. I give this explanation rather fully for, as can be seen, my negligence caused the enquiry to assume a new aspect. Under the conditions I had created the work now resolved itself into a detailed study of the strains isolated from the atarters, and the determining, if possible, of the ability of one or more strains to produce experimentally in cheese the odour and flavour characteristic of the organism in milk. And, as far as "judgment on quality" could decide, the flavour in the experimental cheese approx- imated the "slight-cooked" flavour in the Cheddar cheese to which refer- ence has already been made. EXPERIMENTAL CHE BSE -MAKING. Encouraged by the aroma and flavour produced in milk by certain of the cultures recovered from the starters, one strain from starter "B" recorded as "B37", was cultured in milk and used as a starter in the milk employed for making the experimental cheese. Kingston cheese was selected for the experimental work because of its comparatively small size and rapid ripening qualities, and consequently its suitability for periodical - 6 - examination» The process as employed in our Laboratories (13) was adhered to as strictly as possible, some slight modifications were found necessary however. This arose from the fact that a pure culture of "B37" when used alone as starter caused an exceedingly slow development of acid through- out the cheese-making process. The milk employed in these experiments was pasteurized in the usual manner (13). To facilitate the more rapid production of acid three percent of a freshly clotted and active culture of "537" was employed as starter, the milk was allowed to develop higher acidity than usual before the rennet was added, and later the curd was permitted to sink in the whey for ten minute intervals between stirring. In this maimer sufficient acid developed and the cheese was put to press with the whey testing 0.4 percent lactic acid. The resulting product developed a characteristic Kingston cheese texture. It is interesting that to note at no time during the process was any unusual aroma or flavour noticeable; four days later, however, the "slight-cooked" flavour, which had been first noticed in the Cheddar cheese, was quite evident. This flavour persisted until one month after the date of manufacture and then gradually became obscured by other flavours. THE DETAILED CULTURAL STUDY. The study now commenoed of the strains already isolated from the starters and such strains as were recovered from the experimental cheese. - 7 - MEDIA EMPLOYED. Orla-Jonsen*a Casein Digest Broth (3). 3000 c.c. tap water, 280 gms. commercial acid casein, 8 gms. pepsin and 36 c.c. concentrated hydrochloric acid are filled into large glass Erlenmeyer flasks, which are then plugged; the restating mixture is digested for 10 days at 38° C. The yellow digest, obtained by filtering the mixture at the end of this time, is made up to 2500 c.c. with water and then 10 gms. of di-basic potassium phosphate and 5 gms. of magnes- ium sulphate are added to it. This "double strength" broth is neutralized with a concentrated solution of caustic soda until it is nearly neutralto litmus paper, and then adjusted with N caustic soda until 10 c.c. of the broth requires 1.9 to 2.1 c.c. N caustic soda to neutralize it when phenol- phthalein is usei as indicator. The pH should be slightly more than the usual pH 6.8, for sterilization will increase the hydrogen ion concentration. This "double strength" broth can be sterilized and stocked. Before using for the various media it must be cleared with egg albumin and diluted with an equal volume of water. It can then be used as a broth or in the preparation of agar and gelatin media, it must be sterilized in the final container. Milk. Freshly separated milk is put in flasks and sterilized for three successive days. Chalk Milk (3). Milk as above to which has been added 3 percent of precipi- tated calcium carbonate is sterilized under pressure in the usual manner. Litmus Milk. Milk as above to which enough Azolitmen has been added to give the required blue colour is tubed and sterilized under pressure in the usual manner. Nutrient Agar Gelatin Broth Nitrate Agar Whey Agar Peptonized Milk Agar Difco (Dessicated) (14) Carbohydrates. The purest that could be obtained, principally the products of Difco (14) and some of Khalbaum. METHODS. Total and Volatile Acid Determinations According to the Method of Hammer (5). Chemical Analysis of Chalk Milk Cultures Following the Methods Employed by Orla-Jensen (5). ijg- to 2 litre Erlenmeyer flasks are obtained and into each flask is measured 700 c.o. of fresh separated milk and 3 per- cent (by weight) of precipitated calcium carbonate. The re- sulting mixture is sterilized under pressure in the usual manner, and is then ready for inoculation. A moderately heavy inoculation is desirable; generally 1 c.c. of a freshly clotted milk culture will prove sufficient. The flasks are weighted, the weight recorded, and than they are incubated at a temperature approaching the optimum of the organism being studied for a period of six weeks. A control sample is included with each set. At the conclusion of the incu- bation period analysis is commenced. Observing the usual precautions one drop of each culture is inoculated into a sterile water blank, one c.c. of the resulting dilution is "plated" using appropriate media, and the number of colonies which develop recorded. Also a stained preparation is made from each culture. The foregoing determinations give an index as to the purity of the culture and the degree of longevity of the organism. Each flask is than made up to the original weight with distilled water and subsequently filtered through a filter paper disigned for coarse pre- cipitates. Care is taken to prevent evaporation as the filtering process frequently occupies 12 to 24 hours. The filtrate is used as quickly as possible, for, after re- moval from the chalk and subject to contamination, there is a danger of further fermentation whereby the chemical com- position will alter. The serum thus obtained is retained in tightly stoppered flasks and used in the following de- terminations :- Volatile Acid. 205 c.c. of the serum is placed in a hard glass round bottom distillation flask and 0.5 c.c. of concentrated sulphuric acid added. The whole is mixed and then allaved to stand for 1 hour. The volatile acid is obtained by steam dis- tillation, care being taken to keep the volume of the liquid in the distillation flask constant. The first 900 c.c. of the distillate is collected and titrated with N caustic soda, using phenolphthalein indicator. The OT volatile acid figure is expressed as the number of c.c. of N caustic soda required to neutralize the first 900 c.c. ofTTT the distillate. Total Nitrogen. These determinations are made of the control sample only, and must always be at least in duplicate. The nitrogen is det©mined on 5 c.c. portions of the control milk using the Kjeldahl method.• Soluble Nitrogen. Duplicate determinations are made, 50 c.c. of the original serum are pipetted accurately and run into a 250 c.c. volu- metric flask, 100 c.c. of distilled water are added and then 1 percent (by volume) of glacial acetic acid until a floccu- lency occurrs, 5 c.c. at least are added and as soon as a precipitate is evident one more 5 c.c. lot. Never more than 25 c.c. of acetic acid solution is used. The mixture is heated in a boiling water bath for 30 minutes, cooled, the flask filled to the 250 c.c. mark with distilled water, and then filtered. 50 c.c. of the filtrate is taken for the Kjeldahl* and not more than 20 c.c. of concentrated sulphuric acid is used. Amino Nitrogen. One determination for each culture is usually sufficient. 100 c.c. of the filtrate obtained above, after precipi- tation with acetic acid are taken, 30 c.c. of a 25 percent (by volume) solution of sulphuric acid and 20 c.c. of a 10 percent solution of phosphotungstic acid are added; the mixture is corked and left in the dark for 12 to 24 hours. It is then filtered and 75 c.c. of the filtrate are taken for the Kjeldahl* determination, not more than 15 c.c. of concentrated sulphuric acid is used. * The standard Kjeldahl method employing copper oxide and potassium sulphate was followed throughout. The Soluble and Amino Nitrogen figures obtained for each culture are expressed as a percentage of the total nitrogen of the control sample. - 10 - Determination of Quantitative Acid Produced from Carbohydrates According to Orla-Jensen. (3) The requisite carbohydrate is added to casein digest broth at the rate of 2 percent (by weight) and then dissolved by warming. The broth prepared in this manner is accurately measured in 10 c.c. portions into large test tubes which are then sterilized carefully Tinder 13 lbs. pressure for 25 minutes. The tubes of carbohydrate broth are inoculated from vigorous milk cultures of the organisms under study, and are then incubated, together with a control set, for two weeks. If any cultures appear inactive after 48 hours incubation they must be re-inoculated. At the conclusion of the 2 weeks incubation period 10 drops of a 2 percent solution of phenol- phthalein are added to each tube, and the number of c.c. of an N solution of caustic soda required to neutralize the acid in 4 each tube is determined. The quantity of N caustic soda necessary to neutralize the free acid in the 4 control set is also found. The figure obtained for each control carbohy- drate is subtracted from that found for each culture in the same carbohydrate and the resulting figure is expressed as grams of acid per mille. (As recorded in Table 3). A culture which in any carbohydrate gives -under 1 gram of acid per mille is considered not to ferment that carbohydrate. Determination of Quantitative Acid Produced from Milk. The method followed is identical with that above except that 10 c.c. of sterile separated milk are substituted for the 10 c.c. of carbohydrate broth. The Method for Quantitative Examination of Cheese. This is as employed in Barthel's laboratory. Special hard glass test-tubes, each of which contain a glass rod with a bulb blown at one end, are filled with 9 c.c. water, plugged by means of the glass rod wound with absorbent cotton, and subsequently sterilized. Into each tube one gram of cheese is weighed observing aseptic conditions, an emulsion is made by means of the glass rod, and from the resulting mixture the required dilutions are obtained by the standard method, and plates are poured. - 11 - The eight strains previously isolated from starters, and two strains recovered from the experimental cheese,* were submitted to the detailed cultural study. The results follows- The Influence of Temperature upon Growth. All the isolated strains clotted milk rapidly at 30® C, hut while "B43" grew rapidly at 38®C. and very slowly at 20® G, the remain- ing nine cultures showed little or no variation when carried at 38® C. or at 23® C. Before the quantitative acid produced from various carbohydrates was determined eight strains were grown in 10 c.c. sterile milk tubes in order to find at what temperature they gave the highest percentage of acid. The results,noted in Table 1, show that at three very different temperatures there was no visible variation in the quantity of acid given by any of the strains tested. As a result of the above findings it was decided to employ a temperature of 23® C. for all the cultural work. TABLE I. Lactic Acid in Grams per Mille given by Bight Cultures in Milk Employing Three Different Temperatures of IncubatlonI ~ Culture No. Lactic Acid in Grams per Mille at 20® C. 30® C. 38® C B37 7,2 7.2 7.4 B43 7.0 6.8 7.2 B3 7.4 7.2 7.4 B39 7.4 7.2 7.2 A39 7.9 7.6 7.9 A43 7.2 7.2 7.2 A10 7.0 7.2 7.2 A15 8.1 7.9 8.1 * The isolation of the strains from the experimental cheese is des- cribed fully in Appendix I. Quantitative Estimation of Acid in Carbohydrate Media. In order that the amount of acid produced from carbohydrates might be determined eight strains were selected and subjected to the quantitative estimation of acid. The results are included in Table 3, and since they are of critical importance in classifying strains following Orla-Jensenfs method (3) they will be interpreted later. Reaction to Ordinary Culture Media. •Hie ten cultures being studied were employed, all reactions being determined at 23° C. Milk clotted with a clean acid clot and a re- duction of the litmus. Nitrates were not reduced to nitrites and gelatin was not liquefied after 16 days at 20* C. All the cultures were character- ized by an extremely poor growth on nutrient agar, gelatin, and in nutri- ent broth; when one percent lactose was added to the gelatin there was a marked increase in growth. A plentiful supply of available nitrogenous material, such as casein digest and peptonized milk agar contain, caused a more rapid and heavier growth. Morphology. All the strains were positive reactors to Gram's Stain. The morphology on different media varied, but each culture compared closely in this respect with the Streptococcus cremoris forms shown in Orla- Jensen's photo-micrographs (3). Preparations made from active milk cultures and stained with methylene blue showed that the cells at this stage were surrounded by a large transparent capsule; these capsules were not noticed in older milk cultures. In nutrient broth extremely long, and « I n - frequently intertwined, chains were formed. On agar and gelatin media large, regular, oval cells were fotmd. Such cells generally occurred in pairs or singly, three cultures (B3?, 13 and A39) formed short chains on these media. Total and Volatile Acid Figures Obtained by Hanmer's Method (5). Three cultures were submitted to HammerS determinations for total and volatile acid production. The results of the volatile acid deter- minations are included in Table 2, and compare with certain of the figures obtained by Hammer for Streptococcus lactlcus, but are not nearly as high as those given by the "associated" organisms. The total acidities obtained are in keeping with those given by Hammer's Streptococcus lacticus, and are very much higher than those given by the "associated" organisms. TABLE 2. Volatile and Total Acid Production of Three of the Strains Under Observation (Hammer's Method). Culture Total Acidity Volatile Acidity % (c.c. N Na OH) 10 A10 0.90 3.0 A15 1.06 4.2 B3? 1.01 3.0 - 14 - Volatile Acid Figure by Orla-Jensen'a Method (3). The three cultures examined for volatile acid by Hacmer's method were also subjected to the determination for volatile acid according to Oria-Jensen (3). The findings are given in Table 3. It is to be observed that the volatile acid figure obtained by Orla-Jensen's method is consider- ably higher in each case than is the figure obtained by Hammer's method for the same culture. While strictly speaking the two methods are not comparable, a study will show that they are quite closely rela ted as far as the quan- tities of culture, reagents and distillates taken are concerned. It would appear therefore that in chalk milk there is a greater tendency for these organisms to form by-products, such as volatile acid, than is the case where no chalk is present to neutralize the lactic acid formed. Amount of Soluble and Amino Nitrogen Formed. Preliminary nitrogen determinations, following the method de- vised by Orla-Jensen (3), were preformed using cultures A10, A15 and B37. While the figures obtained were closely similar to those given by many of Orla-Jensen's Streptococcus cremoris (3) they were not considered sufficiently accurate to warrant insertion in Table 3. This is partly attributed to the fact that although satisfactory facilities for the nitrogen determinations were available, such facilities at the time of the investigation could only be obtained in a building some distance from that in which the cultural work was preformed. The practical diffi- culties attendant upon the carrying out cf these determinations in two laboratories rather widely separated must not be overlooked. In a measure, these physical disabilities were partly responsible for the somewhat TABLE 5. I H "" 1 11 Ilk - Ko> Isolated from La ct ic  A ci d R ot ar y Po w e Ni tr og en  So ur ce  — | | £ s # s &H Vo la ti le  A ci  Fi gu re  G ly ce ri ne  X yl os e SS < c e £ 2 £ So rb it e M an ni te  6 >3 Gl uc os e M an no se  G al ac to se  Sa cc lia ro st  I © 1 I ti 111ï  ti D ex tr in  St ar ch  X ' l!1 jl Total N. Sol. N. A.N. f' JJ7 Starter B •555 0.7 0.5 0.9 0.7 0.5 0.? 5.7 4.5 >05 1.9 1 . 4 1.25 3-7 0.9 O.7 0.5 !0.7 0.35 1 7.2 J3 Exp. chee 40 lays o se Ld 0.5 0.7 1.1 0.7 0.2 0.? 5.7 4.3 3.3 1.5 0.9 1.6 3-4 0.9 0.7 0.5 0.5 0.35 1 6.9 AIO Starter A 6.3i 0.5 0.5 0.9 0.5 0.2 0.7 5.05 5.0 4a2 3.7 0.9 0.9 4.3 0.7 O.7 0.5 0.5 0.35 1 7.0 M " A 12.7; 0.9 0.5 1.0 1.0 0.7 o.? 4.5 5 . 3 4.2 2.6 1.1 1.8 4.1 0.7 0.7 0.7 0.7 2.6 1 8.1 AJÛ A m CD 60 0.7 0.7 1.4 0.7 0.5 0.7 5 . 4 5.2 5.2 4.1 1.0 0.8 5.2 0.8 0.5 O.35 0.6 395 1 7.9 tì 1 ü •rH CD m ai Starter B o 0.5 0.2 1 . 4 0.5 0.7 0.9 6.1 5.? 3-3 5.0 0.8 0.9 5-5 0.7 0.7 4.1 0.7 0,6 1 7.4 B43 " B 0.7 0.8 1 . 4 0.6 0.8 0.9 5.5 6.3 5-75 3.6 1-25 5.2 5.5 0.9 0.8 2.4 4.4 3.3 2 7.2 4 Exp. ohee 54 days o se Ld 0.5 6.1 1 . 4 0.5 0.8 o.l 7.2 1.6 4.7 935 1.5 1.9 6.2 6.1 0.5 0.5 0.5 035 1 6.9 § «¡¡y - 15 - erratic nitrogen figures obtained, even though the prescribed methods were faithfully followed. CLASSIFICATION OP THE ORGANISMS STUDIED. Hardly sufficient work has been done to warrant the placing of any of the isolated cultures in the well defined groups given by Orla- Jensen (3). Therefore the nomenclature herein submitted for certain of the strains under study is merely advanced pending further investigation, While the carbohydrate reactions noted in Table 3 have been considered the critical factor relating to the classification of the organisms, the determinations previously outlined, which embody the relation to temperature, cultural characteristics, morphology and to some extent volatile acid and nitrogen figures, have all been given due consider- ation . In order to facilitate the actual classification it was thought advisable to divide the isolated strains into two main groups which are reported as follows Group (a) Included in this group were cultures B37, 13, A10, A15 and A39. These strains were all characterized by their ability to coagulate milk rapidly at 23° C. with the production of a relatively high percentage of lactic acid and with the formation of chains in milk. In their reactions to the various carbohydrates as shown in Table 3, they approached very closely to the true Streptococcus cremorls strains. The sum total of their characteristics would cause them to be placed as Streptococcus cremorls (0rla-Jen3en), the only variation being their ability to impart - 16 - to milk an acrid flavour and a pungent aroma. Of particular interest in this group were cultures 13 and B37, the former being isolated from the experi- mental cheese in the manufacture of which the latter was used as the starter. The sum characteristics of these cultures was identical, the car- bohydrate reactions being almost the same throughout; thus the Postulates of Koch were proved with respect to the experimental cheese. Group (b) Three cultures were placed in this class, namely B3» B43 and 4. Culture B3 differed from the five included in Group (a) only in the fact that it fermented dextrin. Orla-Jensen (3) in speaking of the fermenting powers of Streptococcus cremorls remarks that, " Saccharose is practically not fermented at all, and maltose, and thus also dextrin, only exception- ally to any considerable extent". Since salicin was not fermented and chains were formed in milk this strain could hardly be classified as Streptococcus lactis. It appears therefore that B3 was a dextrin ferment- ing. Streptococcus cremoris strain. It was observed that the strain carried as B43 grew very rapidly at 37® C, while very slow growth in com- parison resulted when the organism was incubated at 20° C. While the mor- phology of this culture was not greatly different from the others, it had been noticed that there was a tendency to form chains having slightly broader segments than the rest of the isolated strains. Now B43 fermented salicin, starch and dextrin but not inulin or raffinose. In a dissertation on the fermenting powers of Streptococcus bovis Orla-Jensen (3) states that it "Is a pronounced starch fermenter and will - with good sources of nitrogen - also ferment inulin and raffinose." When a comparison of the - 17 - sum characteristics of strain B43 was made with those of the typical Streptococcus bovis strains of Orla-Jensen (3) it appeared that it could bes* be classified as a strain of Streptococcus bovis which neither fermented inulin or raffinose. Culture 4 was similar in all its reactions, excepting the fermentation of carbohydrates, to those strains placed in Group (a). While the former fermented xylose and raffinose the latter strains did not ferment these carbohydrates. This strain could not be placed in any of Orla-Jensen*s classes of lactic acid producing strepto- cocci and was arbitrarily classed as xylose and raffinose fermenting Streptococcus cremoris strain. SUMMARY 1, Certain Cheddar cheese manufactured in the Dairy Department of the University of British Columbia developed a flavour which was designated as "slight-cooked". 2. When inoculated into sterile milk pieces of these cheese caused clotting, aM gave a pungent aroma and an acrid flavour. 3. Stained preparations made from the freshly clotted milk showed cocci in chains. 4. From the starters employed in the cheese-making 129 strains were isolated in pare culture, and all clotted milk. Cf the 1?S> strains 113 formed chains in milk. 5. When inoculated into milk certain of the 113 strains gave an aroma and a flavour similar to the aruma and flavour specified under Z, 6. One of the 113 strains, culture B 3?, was used as a starter in the making of experimental cheese. 7. The experimental cheese developed a "slight-cooked" flavour apparently identical with that originally observed in the Cheddar cheese. 8. Of the organisms isolated from the starters and from the ex- perimental cheese, organisms giving the aroma and flavour defined, 8 strains hare been studied in detail. - 19 - 3, Tentatively the 8 strains have 'been classified as follows:- Cultures B37, 13, A10, A15 and A39 as Streptococcus cremoris Orla-Jensen. Cultures B3 and 4, with reservations, as a-typical strains of Streptococcus cremori3 Orla-Jensen. Culture B43 as Streptococcus "bovls Orla-Jensen. 10. Culture 13, one of the organisms isolated from the experimental cheese, proved to oe identical in every characteristic studied with B37, the strain used as the starter for these cheese. 11. Hence; with respect to culture B37 and its relation to the production of cheese with a "slight-cooked" flavour, the Postulates of Koch have been satisfied. - 20 - A C K N O W L E D G M E N T . It is my pleasant duty to thank Professor Wilfrid Sadler who has advised and guided me during the progress of the work, with whom I have from time to time consulted, and who has given me most valuable aid particu- larly in connection with the organisms described. 1 am indebted to The University of British Columbia for appar- atus and facilities which have "been placed at my disposal by the Department of Dairying. - 21 - REFERENCES. (1) Conn, H.W., 1894. "Bacteria in the Dairy". An. Rept. Storrs Agr. Exp. Sta. 7: 57-68. (2) Conn, H.W., 1895. "Bacteria in the Dairy". An. Rept. Storrs Agr. Exp. Sta. 8: 14-40. (3) Orla-Jensen, A., 1919. "The Lactic Acid Bacteria" (in English). With Plates. D, Kgl. Danske Vidensk. Selsk. Skrifter, Haturv. og Mathematisk Afd., 8, Raekke, V. 2. Kobenhavn. (4) Hammer, B.W., and Bailey D.E., 1919. "The Volatile Acid Product-ion of Starters and of Organisms Isolated from Them". Resch. Bull. 55, Agr. Exp. Sta., Ames, Iowa. (5) Hammer, B.W., 1920. "Volatile Acid Production of S. lactieus and the Organisms Associated with it in Starters". Resch. Bull. 63, Agr. Exp, Sta., Ames, Iowa. (6) Hammer, B.W., and Sherwood, P.P., 1923. "The Volatile Acids Produced by Starters and by Organisms Isolated from Than". Resch. Bull. 80, Agr. Exp. Sta.,Ames, Iowa. (7) Orla-Jensen, S., (Trans, by Arup), 1921. "Dairy Bacteriology". p. 118. J. and A. Churchill, London. (8) Sadler, Wilfrid, 1911. "A Eote on an Organism Producing a Burnt Milk Taste". Central, fUr Bakt. 2, Abt. 291, 1-3. (9) Leitch, Renwick H., 1918. "Burnt Flavour in Dairy Products Caused by Streptococcus Lacticus". Bull. West of Scotland. Agr. Col. pp. 21 and 22, Glasgow. (10) Hammer, B.W., and Cordes, W.A., 1921. "Burnt or Caramel Flavour of Dairy Products." Resch. Bull® 68, Agr. Exp. Sta., Ames, Iowa. (11) Sadler, Wilfrid, 1926. "The Production of a "Caramel" Odour and Flavour in Dairy Products by Streptococcus laetis" (Lister). Trans. Roy. Soc. Can Third Series, 20, Sec. 5, 395-408. Ottawa. (12) Hucker, G.J., and Marquardt, J.C., 1926. "The Effect of Certain Lactic Acid Producing Streptococci Upon the Flavour of Cheddar Cheese". Tech. Bull. 117, N.Y. State Agr. Exp. Sta., Geneva, N.Y. M R - 2 2 - (13) Sadler, Wilfrid, 1926. "The Bacterial Flora of Kingston Cheese as shown fcy Micro-photographs". Trans. Roy. Soc. Can. Third Series, 20, Sec. 5. 411-417. (14) Digestive Ferments Co., Detroit, Michigan. - 23 - APPENDIX I ISOLATION OP THE CULTURES PROM THE EXPERIMENTAL CHEESE. Periodical bacteriological examinations of the experimental cheese made using a culture of "B37" as a starter were conducted in order, if possible, to satisfy the Postulates of Koch. An enrichment method proved to be the most satisfactory manner for isolating the organ-isms under study. Sterile milk was employed as the en-richment media; small portions of the experimental cheese being introduced directly into the flasks of milk which were then incubated at 23® C. 37® C. proved to be too high a temperature for satisfactory multiplication of the lactic acid streptococci under study. The milk as it clotted in these flasks was examined to determine the aroma; a stained preparation and qualitative plates using peptonized milk agar were then made, the plates being in-cubated at 23® C. Representative colonies were picked from these plates and inoculated directly into tubes of litmus milk. Cultures so isolated were examined, as soon as the milk clotted, to ascertain if any produced an un-usual aroma and also with the view of determining their morphology. In this manner cultures giving an aroma and flavour in milk similar to that produced by "B37" were isolated when the cheese was twenty, forty and fifty-four days old. None of these types were isolated when th® cheese was three months old. Cultures 13 and 4, isolated when the experimental cheese was forty and fifty-four days old respectively, were retained, as representative types, for further study. In addition to the above qualitative examinations two quantitative examinations were also preformed with the object of determining the frequency of occurrence of the flavour producing streptococci in the cheese. Of forty-one representative colonies isolated from quantitative plates made when the cheese was two months old, none formed chains in milk, and chiefly rod forms were found. Stained prepar-ations were prepared of thirty colonies from the above plates and all proved to be rods. Again at the age of three months an examination of quantitative plates showed twenty-four representative colonies to be rod forms. Unfortunately no examination was made before the cheese was almost three weeks old. It appears from the data obtained as a result of the above examinations that after - 24 - the cheese was three weeks old the flavour producing streptococci were in the minority. The fact that types closely similar to "B37" were isolated when the cheese was almost two months old and that in each case the enrichment milk clotted rapidly with an aroma aod flavour similar to that produced by "B37" in milk, suggested that the "slight-cooked" flavour in the experimental cheese resulted from the action of these micro-organisms. Before any definite statement could De made it was necessary to preform de-tailed cultural work on the isolated strains in order that a comparison might be made between Culture "B37" and those isolated from the experimental cheese.


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