550

LACTIC ACID BACTERIA ASSOCIA.rED WITH RAW PLANT FOOD MATERIAL'

J. OnviN MUNDT Departments of Food Technology ond Microbiology University of Tennessee Knoxville, Tennessee 37916

(Received for puh1ication July 31, 1970)

ABsTRACT f'ration. There are specific sensitivities to the com­ The lactic acid group of bacteria occur on plants with some monly used inhibitors incorponited into selective med­

degree of constancy, but not of consistency, and seldom at ia; not all members will thrive on the same media; Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/12/550/2398486/0022-2747-33_12_550.pdf by guest on 30 September 2021 high levels of population. Their role on the surfaces of plants catalase-negative rods, or sphC'res which may oc­ must be enumerated by a liquid, · MPN technique. cur in pairs, chains, and tetrads. They require car­ Each of the problems has been overcome, and mqst bohydrates for energy, amino acids for nitrogen nu­ of the specific mcthodologies hayie ·been r~corded in trition, and various growth factors. 'Vith only these the literature. properties in common, it is not surprizing that some genera and some specie's within genera can cope with OmciN ON PLANTS the climatic conditions of nature and are found pri­ marily on plants, whereas others are intimately as­ The question of origin of the various lactic acid sociated with a warm-blooded host; and some species bacteria on plants has preoccupied workers during appear to be almost equally at home in either environ­ recent years. Despite intensive study, the problem is ment. generally unresolved. Undoubtedly, many over­ When one examines spontaneous or deliberately winter in soils, although they are seldom isolated induced fermentations, such as those of cabbage, ca­ from this source, and then only if the plant growing cao pods, milk, and a host of other items. or when on that soil or adjacent to it, harbors them. Strep­ one determines the identity of the dominant species tococci have been isolated from the ovules of green on forage grasses, one concludes that relatively few beans and of com, and overwintering in the seed is species of the lactic acid bacteria are associated with a possibility. Tht:y are present in almost astronomical plants. When one goes directly to the plant, a much numbers within the bodies of some insects, such as wider range of speci~s is encountered. Then one con­ members of the Coleoptera and Hymenoptera. There cludes that during fermentations conditions are may be a continual reseeding onto plants from ani­ conducive to the selective enrichment of certain mal wastes on the part of those associated with the species, to the virtual or even real exclusion of the animal body, through the intervention of both in­ remainder. One example will suffice: Streptococcus sects and wind. 'Vhereas fecal origin is an attrac­ faecium var. casseliflavus appears to be more prolific, tive postulate, it is not entirely compatable with the and much more widespread in nature, than is Strep­ distribution patterns of some of the lactic acid bac­ tococcus lactis. It is an exceedingly active bacterium; teria in agronomic and wild environments. it thrives at nature's temperatures; yet it has not ever been reported as an agent in the fermentation of GROWTH ON PLANTS cabbage, cucumbers, or other materials. Lactic acid bacteria possess the potential for

CULTIVATION AND ENUMERATION growth on the surfaces of plants, whether they are present in pure culture or in a naturally competitive The lactic acid bacteria are not difficult to culti­ situation. Several species of these bacteria, S. lactis, vate. Problems arise, however, in attempting enum- Streptococcus faecalis var. liquefaciens, and Lacto­ bacillus plantarum, are recoverable from the stems, arils, and primary and secondary leaves of beans, 'Presented at a Seminar on Spoilage Bacteria, Indicator Or­ cabbage, rye, and com, when the bacteria have been ganisms, and Pathogens in Raw Plant Foods at the 70th Annual Meeting of the American Society for Microbiology, introduced to either control or to sudace-sterilized Boston, l\Iassachusetts, April 26-May 1, 1970. seeds, and these have been planted into either con- LACTIC Acm BACTERIA 551

TABLE l. THE FREQUENCY IN IIECOVERY OF SPHERICAL LACTIC when present, with a range from 1.5 x 10' to 2.6 x AClD BACTERIA FROM 156 PLA.'IT SAMPLES AND THE FREQUENCY w•;g, in a study involving 156 samples of vegetables. AS THE SOLE SPEL'IES RECOVERED Streptococcus faecium var casseliflavus, while often Frequency in recovery Frequency as present in low numbers, has been enumerated at 4 x Bacterium :Number Per cent sole species 10"/g of summer squash peel and of corn flowers, occasionally exceeding the numbers of the ubiquitous Streptococcus faecalis 13 8.3 2 L. mesenteroides. Aerococcus viridans often is present S. faecalis var. liquefaciens 45 28.8 13 Streptococcus faecium 24 15.4 2 in low numbers on succulent plants such as summer S. faeclum var. casseliflavus 44 _28.2 12 squash and greens, but it has not been found on the Leuconostoc mesenteroides 83 53.2 28 more dry vegetable such as the green bean. Aerococcus viridans 11 7.0 0 Streptococci Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/12/550/2398486/0022-2747-33_12_550.pdf by guest on 30 September 2021 TABLE 2. THE IDENTITY AND FREQUENCY IN OCCURENCE OJ," Typical frequency in the occurrence of the genera SPECIES OF LACTOBACILLI RECOVERED FROM THE ORAL CAVITY, and species of the spherical lactic acid bacteria on THE HUMAN INTESTIONAL TRACT AND PLANTS1 vegetables is shown in Table 1. Leuconostoc mesen­ Oral Intestinal teroides is the most frequently recovered species, Species ca\ity tract Plants followed by S. faecium var. casseliflavus and S. fae­ calis var. liquefaciens. Streptococcus faecalis, S. fae­ Lactobacillus acidophaus + + (8)0 Lactobacillus lactis + (5) cium, and A. viridans !lie recovered less frequently, Lactobacillus leichmannli + + (1) in the range o£ 7 to 15% of all samples of vegetables Lactobacillus salivarius + + (1) + (1) included· in the survey. Lactobacillus ~ellabiosus + + {5) The spherical bacteria occur more often as mix­ Lactobacillus casei + + (9) + (3) tures on plants than as the sole species. The figures Lactobacillus plantarum ( 10) + (35) + + in the last column of Table 1 show that during the Lactobacillus fermenti + + (12) + (23) Lactobacillus brevis + + (3) + (21) study in question, S. faecalis, while present on 13 of Lactobacillus buchnerii + + (3) + (1) 156 samples, was the sole species obtained from only Lactobacillus viridescens + (1) 2 ·samples. Leuconostoc was the only species re­ covered from 28 of the 83 samples from which it was "Frequency in recovery isolated. The simultaneous occurrence of 3, 4, or 'Data from three references at end of paper. even 5 species on the same plant is not unusual. The lactic acid bacteria .do not thrive on plants trol or heat-sterilized garden soils. Estimated in­ during the cold month~ of the year. They are re­ creases in numbers on the plant over those estimated covered rarely from dormant and overwintering to have been introduced to the seeds range from a low plants, and with less frequency from non-succulent of 40-fold to a high of 750-fold. plant parts such as leaves, than they are from flowers The levels of population which the several lactic and fruiting structures. Streptococcus faecium var. acid bacteria achieve on plant surfaces undoubtedly casseliflavus prefers the cooler months during spring are restricted severely by the amount of available nu­ and early summer. It is obtained only infrequently, trients and also by competition with the less de­ and then usually in low numbers per unit weight of manding, usually Gram-negative, bacteria. Sterilized tissue, after mid-July. On the other hand, a direct washing and conveying waters used in vegetable correlation exists between the advance of the sum­ processing have supported the growth of selected mer season and the frequency in recovery of S. fae­ species of the lactic acid bacteria to populations in calis var. liquefaciens. The increases in recovery are excess of 1 x 107 /ml, provided such waters contained correlated with increasing average daily temperature 0.01 to 0.02% nitrogenous substances. It is felt that beginning with the first isolations in June to mid­ these waters reflect reasonably the adequacy of the September, after which an abrupt decrease in both food material available on the surfaces of plants. incidence and numbers occurs. The occurrence of of lactobacilli and pediococci on plants also seems to Kr:Nns oF PLANTs be correlated to the warmer temperatures, although too few isolations have been made to establish a Relatively few species of 'the lactic acid bacteria firm pattern. Pediococci are not isolated from plants are found ronsisfeatly on -plants, and even these are (in Tennessee) until the end of May, when small not mvariably present. The numbers per gram of grains are in the milk stage. plant tissue ar'e highly variable. The average popu­ Reproduction and the frequency in occurrence of lations of S. faecalis, S. faecalis var. liquefaciens, and the lactic acid bacteria is influenced markedly by S. faecium are less than 1 x 10"/g of plant tissues, rainfall and relative humidity. During one study, 552 LACTIC ACID BACTERIA these bacteria were obtained from all samples of raw, succulent commodities like peas are held too selected vegetables with an average population of long in vats prior to blanching. ·' . 5 1 X 10 /g tissue. During the following year, when The succulent plant seems to be the reservoir of near drought conditions prevailed, these bacteria were A. viridans. It has been isolated also from processing obtained from only two-thirds the samples, with total eqnipment and from spontaneously fermenting vege­ populations at 10% tho~e of the preceding year. tables in holding situations. but not, however, as the The majority of the strongly reducing enterococci dominant agent of deterioration. produce a soft, rennin-like curd which is usually Figures giving the incidence and levels of popuJa­ digested rapidly in stratiform fashion, in contrast tion of S. lactis on plants may be deceptive, because with the hard curd which is digested vertically. many strains of this species are sensitive to the so­ characteristic of the acid-proteolytic streptococci. dium azide incorporated into selective culture media.

The amount of rennin which cultures of each type Hirsch has postulated that S. lactis as it is known to Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/12/550/2398486/0022-2747-33_12_550.pdf by guest on 30 September 2021 produces is very nearly alike; differences in the na­ the dairy bacteriologist is a recently evolved, highly ture of the curd and in typ~ of digestion are at­ specialized membn of a much wider complex. Cur­ tributed to differences in rate of fermentation of rent work in our laboratory indicates this to be true. lactose. The majority of the S. faecalis-like strepto­ In nature, neither lactose fermentation nor deamina­ cocci isolated from Mammalia other than humans and tion of arginine appear to be of paramount value. Dif­ domesticated animals, Reptilia, and plants growing in ferentiation through the ability to grow in broth either agronomic or wild environments, are of the with 4%, but not 6.5%, NaCl, appears questionable; soft curd-producing type. Many of these are also (3- we are able to train many cultures to growth in broth hemolytic. but this property has little differential with 6.5% NaCl with relative ease. Members of the value in the plant setting. wider complex, frequently those unable to ferment Streptococcus faecium is widely distributed in the lactose, have been observed in frozen vegetables by plant environment. It appears to be the dominant workers in England and in the United States. enterococcus in the intestinal tract of boars and of Lactobacilli hogs. For many years, it was difficult to reconcik Studies of the lactobacilli associated with fresh the pro;1erties of cultures of this species taken from plant material are few. There is a popular concep­ plants with the description given for the species by tion that certain species are "plant" types, others are Orlu-Jensm. The recognition of the variant. S. fac­ "dairy" types, and still others are "animal" types. The cium var. casseliflavus, has reduced much of the con­ conception probably arose, becaus3 specific prop­ fusion. The variant is a very active bacterium with erties and selective environments have given prefer­ properties of both S. faecnlis and of S. faecium. It ence to species most adaptable to that environment. ferments many more sugars than do either of these Th~ ability to ferment lactose, for example, enables species. Most of the cultures are motile. It produces milk and its products to serve as enrichment agencies a waterw1uble, pale lemon yellow rigment in the for Lactobacillus casei. cell waH. Especially when first isolated, it is fre­ A list of the species of the lactobacilli isolated quently deformed and appears as a swollen rod. from the human mouth, the intestinal tract of man, Cells of many strains appear to produce buds, and and from plants is presented in Table 2. The figures these give rise to crooked, rather than to straight in the second column indicate the frequency with chains of cells. The variant colonizes on plants very which each species was isolated from 21 healthy hu­ readily, and except for Leuconostoc it is the most mans, and those in the third column the frequency in common of the spherical bacteria on plants during isolation from 160 plants. The most interesting fea­ late spring and early summer. Despite its abundance, ture of the data is the frequency with which the we have not observed it as a primary agent during the plant and dairy species were isolated from the nor­ deterioration of vegetables in holding situations. mal human intestinal contents: Lactobacillus fer­ Species of Leuconostoc other than L. mesenteroides menti, L. plantarum, L. casei, and Lactobacillus brev­ have been encountered extremely rarely in approxi­ is exceeded Lactobacillus acidophilus in the frequency mately 5,000 identifiE-d cultures. As others have re­ of recovery. Several of the plant species have been ported, about 70% of the cultures produce d~xtran implanted into the human intestinal tract by inges­ on sucrose agar. The remainder are detected most tion. In view of the low numbers of lactobacilli on easily through the use of the Gihson-Abd-El-Malek plants, quite oftlim less than 10/g tissue, the postu­ gas entrapment technique. Carbon dioxid':) does not lated ternary cycle, from intestinal discharge to na­ accumulate in the frequently used Smith or Dur­ ture to reimplantation via the oral cavity appears ham tubes,· because of its high degree of solubility tenable. in water. Leuconostoc becomes a problem when There is no reason to believe that one survey, LACTic Acm BAc.'TERIA 553 conducted in a limited geographic area, expresses sources to the plant are as yet unknown. It is definitively the range of species' or their numbers questionable as to whether there is a reseeding from occurring on plants. Plants grown in more temperate animal waste, since we have been able to isolate climates may be the natural habitat for several them only infrequently from dairy cattle fed corn sil­ mesophilic species such as Lactobacillus frigidus, age, but not from the intestinal discharges of poultry, Lactobacillus malefermentans, and Lactobacillus par­ beef cattle, sheep, or hogs. vis, which have been isolated from beers. A recently The genus Pediococcus includes three diverse rediscovered sp::cies, Lactobacillus intermedius, initi­ species groups: those found in beers, typified by ally isolated from wines low in acid, occurs as fre­ Pediococcus cerevisiae, the halophilic Pediococcus quently as any species of Lactobacillus on plants in which is encountered extensively in the Orient, and Tennessee. the plant group. Only members of the latter group, Lactobacilli are able to colonize in the sludges of Pediococcus acidilacti and Pediococcus pentosaceus, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/33/12/550/2398486/0022-2747-33_12_550.pdf by guest on 30 September 2021 expressed vegetable juices on processing equipment. have been isolated from plants. Both species ac­ They can be enamerated in terms af millions per gram company Leucorwstoc, the streptococci, and aero­ of sludge at sites such as the carton filler and the cocei on fermenting wet vegetables prior to blanch­ gate valve at the end of prolonged packing opera­ ing. tions. Pediococci REFERENCES With an overall incidence of 8.7% on plants in our 1. Davis, G, H. G. 1960. Lactobacilli of the human studies, pediococci ar6 not numerous, with a record­ mouth, J. Appl. Bacterial. 22:350-359. ed maximum of 23/g fresh plant tissue. There is 2. Lerche, M., and G. Reuter. 1962. Das Vorkommen no evidence at this time that they colonize on plants; aerob wachsender gram positive Stabchen des Genus Lacto­ bacillus Beijerinck im Darmhald erwaehsender Menschen. they grow, however, at the expense of expressed Zentralbl. Bakteriol. Parasitenk., Orig. 185:446-481. juices on harvesting equipment and in masses of 3. Mundt, J. 0., and J. L. Hanuner. 1968. Lactobacilli fermenting materials. The primary habitats and on plants. Appl. Microbial. 16:1326-1330.

CONTAMINATION OF DAIRY FEEDS BY AIR POLLUTION SAMUEL B. Cuss Veterinaru Science Extension The Pennsylvania State University University Park, Pennsylvania 16802 (Received for publication August 12, 1970)

ABsTRACT has presented serious problems in the. state. There Air pollution of forage for dalry eattle is already a serious have been problems associated with application of problem where industry is encroaching upon land used for herbicides and pesticides which ,have caused major production of feed and forage crops. In Penmylvania, lead herd disasters, but the purpose of this paper is to poisoning and serious metabolic disease . resulting from lime discuss problems caused directly by industrial air plant .stack effluents have caused losses on dalry and beef pollutants. cattle farms. Evidence of intoxication and other metabolic disturbances in cattle in areas close to industries warrants search of pastures or stored forage crops for toxic air pollu­ LIME tants. Pasture areas close to heavily travelled highways may contain appreciable quantities of lead. This situation appears Lime plants have been a major source of air pollu­ to justify inunediate concern by health and agriculture regu­ tion for many years in Pennsylvania and we have ex­ latory agencies. perienced severe nutritional problems in herds graz­ ing pastures or consuming hay or ensilage from land Growth of cities and industry in Pennsylvania con­ in close proximity to lime plants. Nutritionists gen­ tinues to present problems for those who manage erally agree that calcium-phosphorus ratios for dairy livestock and poultry enterprises. Water pollution cattle should approximate 1.5:1. It is virtually im- of surface streams and ground water is· nothing new. In recent years, air pollution contamination of feeds Continued On Page 561