], Milk Food Techrwl., Vol. 36, No. 6 (1973) 317

FATE OF CEREUS IN CULTURED AND DIRECT ACIDIFIED SKIMMILK AND CHEDDAR CHEESE' E. M. MIKOLAJCIK, J. w. KEA:RNEY, AND T. KrusTOFFERSEN Department of Food Science and Nutrition Ohio Agricultural Research and Development Center, Columbus 43210 (Received for publication December 4, 1972)

ABSTIIACT METIIODS Downloaded from http://meridian.allenpress.com/jfp/article-pdf/36/6/317/2399432/0022-2747-36_6_317.pdf by guest on 01 October 2021 Germination, growth, sporulation, anP. survival of Bacillus Bacillus cereus 1, originally isolated from a raw milk sup­ cereus 7 was determined in <:ulturf.-d ( Streptococcus lactis ply, (7, 9) was used. As described previously (1, 9), ClO) and direct acidified (lactic acid) skimmilks. For cul­ were prepared by the non-heat method and vegetative cells tured systems, B. cereus increased initially at approximately in exporumtial growth and synchronous divi.1JOre cotullf:s remained unchanged. Media (Galloway-West Co., Fond du Lac, Wise.) recon­ B. cereus organisms did not influence acid production or stituted to 11% total solids and sterilized at 121 C for 10 multiplication of tho lactic streptococci. In direct acidified min. Cultnred skimmilk was prepared. by inoGUlation at the skimmilk, germination and outgrowth and vegetative C.5!ll level with Streptococcu.Y ku:tts C10. For the direct cell multiplication det:reased as the pH of the system was acidified samples, the pH of the autoclaved skimmilk was lowered from 6.5 to 5.0. In skimmilk at pH 5.0, vegetative adjusted with sterile lactic acid and incubated at 30° C. ce11s failed to multiply and spore germina,tion ceased. In Cheddar cheese wa~ manufactured in the conventional man­ Cheddar cheese manufacture, B. cereus multiplied r-apidly ner from milk pasteurized at 72.2 C for 17 sec and inocu­ during tbe period from the end of to milling of the lated with 1% Hansen's H-4 mixed culture. Two separate curd. B. cereus survived in the spore state in Cheddar cheese trials were made. For each trial, two lots of cheese were during 52 weeks curing. prepared: one was artificially infected with heat-shocked ( 80 C - 12 min) B. cereus 7 spores to yield a final spore It is becoming increasingly evident that Bacillus count of approximately 4,000/ml of milk and the other lot cereus can be an etiological agent in some cases of served as the control. food poisoning. Although to date, the actual mech­ The -egg yolk-polymyxin ( MYP) medium of Mos anism ( s) hy which this occurs is unclear (3 ). sel et al. (10) was used to enumerate B •. cereus organisms in the presence of lactic streptococci. This medium has been In some aspects, B. cereus food poisoning resembles evaluated and utilized by Kim and Goepfert (5, 6) for studies that of perfringens (3, 11 ). There is con­ of B. cereus contamination of food products. Characteristic vincing proof that food poironing by C. perfringens colonies appearing on MYP agar vvere counted following in­ requires consumption of foods containing large num­ cubation of plates at 35 C for 24 hr. In systems rontaining only B. cereus, Standard Plate Count agar was employed. bers of vegetative cells (4). For B. iJereus food poi­ Spore counts were done oo heated samples (80 C 12 min) soning, col.lillts in excess of 10"-10" /g have been en­ using Standard Plate Connt agar containing 0.1% soluble countered in suspeot foods (3). starch with incubation at 35 C. B. cereus is wid3ly distributed. In our 1961 sur­ To enumemte lactic streptocoeei in the presence of B. cereus, vey of raw milk from Ohio farms, B. cereus spores Standard Plate Count agar was used and counts were cor­ rected by substrating B. cereus counts of the same system ob­ were present in 37% of 287 samples examined (7). tained on MYP agar. Higher values were reported by European workers Percent germination, generation time, and number of gen­ (3) who found that over 70% of the milk samples erations were calculated as previously reported (9). contained B. cereus. Dried skimmilk and pasteurized Curd samples for microbiological analy~>is. were prepared by grinding 11 g of curd with 99 ml of sterile 2% sodiun1 milk have been found to be contaminated with B. citrate at 40 C in a Waring Blender. cereus (3, 6). Titratable acidity expressed as percent lactic acid was As part ·of a continuing investigation, we have re­ determined by standard procedures utilizing 0.1 N NaOH and ported on B. cereus spore germination and outgrowth phenolphthalein indicator. (9), proteolytic activity (1, 2), and heat resistance (8) in skimmilk. This study reports on the behavior of REsULTS AND DISCUSSION B. cereus in cultured and direct acidified skimmilk Cultured skimmilk and during Cheddar. cheese manufacture and curing. The effect of lactic acid streptococci on the rate of 'Approved as Jonrnal Series Article No. 111-72 of the Ohio outgrowth and germination of Bacillus spores in skim­ Agricultural Research and Development Center. milk was determined (Fig. 1). During the first 4 318 MIKOLAJCIK ET AL.

With respect to spore outgrowth (Fig. 3) particu­ 7 c::J larly during the period of 4 to 8 hr incubation, the population level and rate of growth varied with the f!.CEREUS / pH of the skimmilk. As the pH decreased from 6.55 to 5.03, there was a corresponding decrease in the ./c5/\ MYP .. - rate of growth and/or in the total count of organisms present at each time interval. ;::::?' 0 \ Spore germination was also influenced by pH of the sysrtem (Fig. 3). With the exception of pH 5.03, f~ in all other instances, spore numbers decreased gradu­ t· ally with increasing incubation indicating germination. + LACTIS l-0::::::::::::;,_-8~EREUS ~· The rate and extent of germination increased with

increases in pH. For example, at 6 hr of incubation, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/36/6/317/2399432/0022-2747-36_6_317.pdf by guest on 01 October 2021 • I the percent germination ranged from 0 for pH 5.03 (77%G)• • ~ • I skimmilk to 97 for pH 6.55 skimmilk. In the later >­ SPORE :E2 I milk, initiation of sporulation was detected at 8 hr. (!) • • Results shown in Fig. 4 are for vegetative B. cereus 0 ·------(97% G) ...... 1 / cells in exponential growth added to direct acidified CEREUS J! skimmilk. Active cell multiplication occurred. in all but pH 5.03 skimmilk. As the pH of the skimmilk was decreased to 5.03, there was a corresponding de­ 0~----~------~------crease in the rate and extent of multiplication of o z 4 6 8 vegetative cells. At pH 5.03, vegetative cell numbers INCUBATION TIME- HOURS- 30°C decreased rapidly indicating that the organisms failed to survive in the acid environment. Figure l. Effect of S. lactis ClO on the germination and outgrowth of B. cereus 7 spores in sldmmiik. The percent For B. cereus :in a direot acidified system, pH 5.0 germination is shown at 4 hr. {Average of 3 trials). appears to be the pH at which inhibition of spore germination and outgrowth and active cell multi­ plication is manifested. However, spores are not hr of incubation, outgrowth of B. cereus was approxi­ destroyed at this pH. In associated growth systems, mately the same whether or not S. lactis was present. as was observed with the lactic streptococci, some Beyond 6 hr, counts of B. cereus in the cultured sys­ tem dropped rapidly, whereas counts of the control milk continued to increase at an exponential rate. At 4 hr, 77% of the B. cereus spores had germinated in the cultured system as compared to 97% for the 0 control. The rate of acid development by S. lactis ClO was not affected by the presence of the B. cereus organ­ §:CEREUS+.§: LACTIS ...... - isms (Fig. 2). In fact, during the period of 6 to 8 br, acid production by S. lactis ClO was slightly high­ er in the presence than in the absence of B. cereus. However, the increased acidic environment did de­ crease markedly the survival of B. cereus cells (Fig. 1). Although not shown, studies with B. cereus 7 spores and cremoris US3 or Hansen's H-4 lJJ yielded similar results. (/)

from a low of 690/g at cutting. 7 Part of the observed increases in counts may have resulted from physical entrapment .of the organisms .....1 in the curd coupled with concentration of the curd :!:6 upon expulsion of the whey...... 'z In one-day old cheese, B. cereus counts on MYP ::J agar wer:e 4,300/g, whereas spore counts of the same gs system were 5,600/g. Apparently, the B. cereus organisms survive in the spore state. MYP agar does not differentiate betwoon the vegetative or spore state, enumerating both states. The difference be­ tween MYP counts and spore counts is probably re­ lated to normal variation in plating techniques and/or to an inability of some of the spores to germinate and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/36/6/317/2399432/0022-2747-36_6_317.pdf by guest on 01 October 2021 outgrow on the selective agar. Spores of species other than B. cereus were not a factor in the increased I 2 spore count because the control lot of cheese pre­ (!) 0 pared without added spores had counts of only 155/g .....1 o- o TOTAL COUNTS at 1 day . •-• SPORE COUNTS As the curing time progressed from 8 to 52 weeks, counts on MYP agar and spore counts varied only slightly from each other and remained relatively con­ 0 stant within the range of 5,500 to 8,200/g. o 2 4 6 8 The flavor, body, and texture quality of both the INCUBATION TIME- HOURS AT 30°C infected and ccmtrol lots of cheese were similar and Figure 3. Effect of pH of skimmilk on germination and highly acceptable. At 52 weeks, the cheese was outgrowth of B. cereus 7 spores. The percent germination is shown at 6 hr. and the pH of the skimmilk in the extreme right column. (Average of 3 trials) TABLE 1. FATE OF Bacillus cereus SPORES L"' THE MANUFACTURE AND CURING OF CHEDDAB CHEESE" Sample Total c!>unt• inhibition of spore germination and active cell multi­ (No./g or ml) plication may occur at higher pH. Milk used for cheese manufacture 120 28 Upon addition of B. cereus spores 3,600 3,900 Cheddar cheese End of ripening 21,500 440 To ascertain the fate of the B. cereus spore during End of renneting 6,000 . 380 manufacture and curing of Cheddar cheese, milk was At cutting: Whey 1,800 30 infected with approximately 4,000 heat~shocked Curd 19,500 690 spores/ml prior to manufacture. B. cereus counts on End of cooking: Whey 2,700 30 Curd 44,000 2,600 selective MYP agar and on Standard Pla:te Count agar Curd at milling 2.20,000 5,600 were done at each step of the manufacturing process Curd at hooping 42,000 6,000 and at selected time intervals during curing at 4 C Cheese after curing for - 1 day 4,300 5,600 for 52 weeks. 7 days 5,500 4,200 14 days 7,000 6,700 For the data shown in Table 1, the approximate 28 days 6,100 5,500 time for each of the following cheesemaking steps 8 weeks 6,000 5,800 was 1 1/4 hr for ripening and renneting, 1 hr for 12 weeks 7,400 7,100 cooking, and 2 1/2 hr for cheddaring. The total time 16 weeks 5,500 5,700 24 weeks 8,200 5,900 from the addition of the spores to hooping was about 30 weeks 6,500 5,700 6 hr. 52 weeks 5,800 6,200 B. cereus com;tts increa~ed approximately 5-fold "Averages of two separate lots of cheese. Control cheeses pre­ during ripening of the milk with a 10-fold decrease pared with the same mllks had 28 spores/ml in cheese milk in &pore counts. At cutting, the curd contained 19,500 and the finished cheese had 155, 180, 58, and 80 spores/ gram B. cereus/g and at milling, the counts had increased following curing of 24 hr, 12 weeks, 24 weeks, and 52 weeks, respectively. to 220,000/g. Following salting and at hooping, the •Total B. cereus count on MYP selective agar. MYP counts had decreased to 42,000/ g. However, "Counts obtained following heat treatment of sample at 80 spore counts of the curd had increased to 6,000/g C- 12 min. 320 MIKOLAJCIK ET AL.

Few or no vegetative B. cereus cells were present in 8 the cheese during the curing period substantiating the finding from the cultured and direct acidified study that the pH of properly prepared Cheddar cheese is inhibitory. It should be noted that if the mechanisms for food ..J6 poisoning by B. cereus involves the pre-formed , :E ..... thm the ability of the organisms to multiply prior to 1- development of sufficient acid by the lactic strepto­ z cocci should be of concern.

REFERENCES

1. Choudhery, A. K., and E. M. Mikolajcik. 1971. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/36/6/317/2399432/0022-2747-36_6_317.pdf by guest on 01 October 2021 in milk. 2. Sweet curd formation. J. Dairy Sci. 54:321- 325. 2. Choudhery, A. K., and E. M. Mikolajcik. 1970. Acti­ vity of Badl!u.Y cereus proteinases in milk. J. Dairy Sci. 53:363-366. 2 3. Goepfert, J. M., W. M. Spira, and H. U. Kim. 1972. Bacillu.1 cereU