506

Journal of Food Protection, Vol. 46, No.6 Pages 506-509 (June 1983) Copynght~, International Association of Milk, Food. and Environmental SanHarians

Factors Influencing the Agaritine Content in Cultivated , Agaricus bisporus

J. J. SPERONI/ R. B. BEELMAN,.· and L. C. SCHISLER2

Departments ofFood Science and Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 16802 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/46/6/506/1655910/0362-028x-46_6_506.pdf by guest on 28 September 2021 (Received for publication December 10, 1982)

ABSTRACT with the proper nutrients needed for growth. The compost temperature rises dramatically during fermentation (as high Agaritine concentrations were determined in fresh mushrooms as 180°F) and serves as a form of compost pasteurization. grown from various spawn strains on several compost types and Concomitantly, compo sting must also either produce sub­ har:ested at different phases of the cropping cycle. A wild spawn stram produced mushrooms with approximately two times the stances that inhibit the growth of competitor organisms or agaritine content of seven other more commercially important remove nutrients needed for their growth. com­ types. Mushrooms harvested from a synthetic compost produced post types are commonly classified according to the starter significantly higher amounts of agaritine than five other compost material used. "Natural composts" consist mainly of types. Additionally, mushrooms harvested later in the cropping wheat straw-bedded horse manure. They are generally the cycle were more likely to have higher agaritine levels compared most widely used and least expensive compost types. to earlier harvested mushrooms. Agaritine was also present in the "Synthetic composts" consist largely of hay and crushed mycelium of Agaricus bisporus growing in liquid culture, but at com cobs and are generally any composts where horse ma­ much lower levels than present in the fruiting bodies. nure is not the major ingredient. "Blended composts" are made from mixtures of "natural" and "synthetic" com­ posts. Additionally, most composts also require additional Agaritine «(3-N-('y-L-( + )-glutamyl)-4-hydroxymethyl­ nitrogen supplements, gypsum to aid in pH control, and ) is a phenylhydrazine amino acid deriva­ often many other ingredients. tive isolated from the fruiting bodies of the commercial Spawning refers to the inoculation of the compost with mushroom (Agaricus bisporus) and several other species the mycelium of a selected strain of Agaricus bisporus. from the same genus (9). Few compounds containing the The strain or isolate selected for use at this phase of the functional group are known to exist naturally, al­ production schedule has a tremendous influence on the though linatine (7) isolated from flaxseed, from phenotypic attributes of the harvested mushroom. For ex­ the false morel mushroom (14), and the compound hyd­ ample, strains may be white, brown or cream in color, razine from tobacco (10) are notable exceptions. have scaly or smooth caps, and display various combina­ Several synthetic hydrazine compounds have been dem­ tions of each. There are numerous strains currently avail­ onstrated to possess carcinogenic potential (13,17,18,22). able to the commercial grower. To date, agaritine has never been shown in direct feeding During the cropping or harvesting period, a very inter­ trials to be carcinogenic, although agaritine analogs have esting and agriculturally unique event occurs. Rhythmic 3- displayed this potential (17,20,21). Toth (19) has exten­ to 5-d harvests, which constitute a single flush or break, siv~ly reviewed the literature concerning mushroom hyd­ are followed by periods when few or no mushrooms are razmes and their toxicological implications. available for harvest. Growers usually harvest from at least In the United States, commercial mushroom growing en­ three but generally no greater than eight flushes per crop tails several production steps which may affect agaritine before aborting the mushroom bed. Hayes (3,4) presented concentration in the harvested mushroom. Three of these an extensive review of the biology and nutrition of steps, i.e., composting, spawning, and cropping, are espe­ Agaricus bisporus and Wuest et al. (23) reviewed the com­ cially critical and are considered in this paper. mercial operations used for its production. Mushroom composting involves a type of fermentation The purpose of this study was to evaluate several of the whereby the original starting material is chemically and production steps that may affect the concentration of biologically altered to provide the mushroom mycelium agaritine in the cultivated mushroom. More specifically, IDepartment of Food Science. this study was designed to determine the following in rela­ 2 Department of Plant Pathology. tion to agaritine concentrations in the mushroom: (a) the

JOURNAL OF FOOD PROTECTION. VOL. 46, JUNE 1983 AGARITINE CONTENT IN MUSHROOMS 507

TABLE 1. Compost study: Precomposting ingredients of selected composting treatments used to grow mushrooms (Agaricus bisporus,

Precomposting treatment No. ingredients (Ibs) 2 3 4 5 6 Horse manure 375 337 180 180 Hay -a 90 117 234 180 Crushed corncobs 90 180 Brewers grain 30 35 25 15 25 Poultry manure 30 30 25 10 25 Potash 2.25 2.25 4.5 4.5 Pelleted corncobs 34 64 130 15 15 15 15 15 15 Total (dry weight basis) 450.00 451.00 427.25 403.25 383.50 429.5

Total 6.90 6.84 6.63 6.28 6.26 6.92 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/46/6/506/1655910/0362-028x-46_6_506.pdf by guest on 28 September 2021 aBlank spaces used to indicate absence of ingredient from that compost type. bAs determined by KjeJdahl method. influence of various composting treatments (compost The strains were randomly distributed among 32 trays (4 trays per strain). study); (b) the influence of various spawn strains of The description of the various isolates used is listed in Table 3. Mus­ Agaricus bisporus (strain study); and (c) the influence of hrooms from five different harvest dates, rather than from peak days of flushes, were used for agaritine analysis since the various strains yielded flushes or breaks during the cropping cycle (flush study). highly variable peak harvest days. In a separate study, the agaritine concentrations of several strains growing in mycelial liquid culture were estimated Mycelial culture (mycelium study). Mycelial cultures of PSt:' strains 324, 352, 351, 310 and 265 were pre­ pared by D. J. Royse using 250-ml Erlenmeyer flasks containing 100 ml of a potato dextrose yeast (PDY) broth (5). The cultures were allowed to ~o 2- grow for 5 wk at 23°C before the mycelium was harvested for agaritine analysis. 25 Sample preparation In the compost and strain studies, mushrooms were harvested from the ~ 20 4- trays comprising treatments and combined into treatment lots for selected flushes and harvest days, respectively. Approximately 200 to 300 g of the ~- .c~ 15 mushrooms (unstretched veils, 1 to 2 in. in diameter) were randomly ::;:"::I selected from each lot, brushed to remove dirt, weighed, placed in '0 polyethylene bags and immediately stored at -12"C. These samples were ." 10 5 iii subsequently freeze-dried. stored in desiccators over caSo4 and analyzed :.:: for dry matter percentage. In the mycelium study, harvesting was done by gravity filtering the liq­ uid media through Whatman No. I filter paper. The retained mycelium was washed three times with 5 ml of distilled water, scraped from the fil­ ter paper into lO-mJ glass vials and immediately stored at -IS"C. The mycelium was freeze-dried and dry weights were determined after Harvest Day equilibrating in a CaS04 desiccator.

Figure 1. Cropping cycle (average daily production yields) of Agaritine analysis Agaricus bisporus PSU-324 strain. Data are averages obtained In the compost and strain studies, agaritine analysis was performed as from six compost types. Numbers with asterisks indicate the peak previously described (15). For the mycelium study, the freeze-dried day of each flush on which mushrooms were harvested for mycelium was pulverized with the aid of a frozen mortar and pestle in 25 agaritine analysis in the compost and flush studies. mI of methanol (Burdick and Jackson, HPLC grade) plus I ml of 1% sodium bisulfite and then extracted for 45 min at 5°C. The extract was MATERIALS Asn METHODS gravity-filtered through Whatman No.1 filter paper, the retentate washed with methanol (10 ml) and the filtrate collected in a l25-m1 rotary Mushroom culture evaporator flask. The extract was taken to dryness and resuspended in 4 In the compost study, cultivated mushrooms, Agaricus bisporus mI of 0.005 N NaH2P04 at pH 4.25. After this step. the sample handling Pennsylvania State University Culture Collection strain 324 (PSU-324), and chromatographic procedures were essentially as previously described were grown at the Mushroom Research Center (MRC) (Department of (15). Plant Pathology, University Park, PAl. The mushroom production system was similar to that described by Beelman et aI. (1) with six composting Data analysis treatments randomly distributed among 48 trays (8 trays per treatment). In both the compost and flush studies, a 2-way analysis of variance The precomposting ingredients used for each compost treatment are listed (AOY) was employed. In the compost study, the various compost mix­ in Table 1. Mushrooms were harvested for agaritine analysis on the peak tures were used as treatments with the crop cycle flushes or harvest dates days of flush numbers 2, 3, 4 and 6 (Fig. 1). as blocks. Conversely, in the flush study, the data from the compost study In the strain study, eight strains of Agaricus bisparus were grown at the were arranged to generate an additional AOY; flushes were considered as MRC in a natural compost similar to compost #1 described in Table I. treatments and compost type as blocks. For the strain study, a I-way

JOURNAL OF FOOD PROTECTION, YOL. 46, JUNE 1983 508 SPERONI, BEELMAN AND SCHISLER

TABLE 2. Compost study: Agaritine concentrations (g!100 g dry weight) in fresh mushrooms (Agaricus bisporus, PSU-324) grown from composts varying in precomposting ingredients as monitored according to the cropping cycle flush number. Compost Flush No. treatment No." 2 3 4 6 Mean .------_.... 1 (Natural) 0.047b 0.261 0.422 0.355 0.273 N 2 (Natural) 0.045 0.171 0.410 0.371 0.236 A 3 (Blend) 0.036 0.129 0.266 0.628 0.265 A 4 (Blend) 0.042 0.228 0.374 0.585 0.307 A 5 (Synthetic) 0.073 0.294 0.530 0.739 0.409 A 1.275 0.746 B "See Table 1 for precomposting ingredients. blndividual values represent the average of two determinations. cMeans followed by the same letters are not significantly different at the P = 0.01 level. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/46/6/506/1655910/0362-028x-46_6_506.pdf by guest on 28 September 2021

AOV was employed as it was not possible to separate the flush variation from the error term. In all cases, Duncan's new multiple range test was I n used to separate treatment means (16), In the mycelium study, no statisti­ 0,7 0.7 cal evaluation was performed. B

RESULTS AND DISCUSSION ::: 0.6 0.6 .s::; CI '0) B N Comparing mean values from the results of the compost 3 E >0 0.5 0.5 study (Table 2) indicated that mushrooms grown in syn­ .... N '0 I'- r<1 thetic composts had higher agaritine levels than mus­ d hrooms obtained from natural or blended composts. Treat­ g 0.4 ...... 0.4 CI ment 6 was significantly higher in agaritine than the re­ Q B CD maining compost types at the P=O.Ol level, whereas treat­ CI c: CD 0.3 ::.... 0,3 B ment 5 could also be separated from the remaining com­ c: A 0 :;: CI '':: <.( posts at the P = 0.10 level. Within a given flush, the mus­ o hrooms harvested from synthetic composts were always if 0.2 0.2 higher in agaritine concentration than mushrooms from natural (Treatments No. I and 2) or blended composts A 0.1 0.1 A (Treatments No.3 and 4), which were statistically similar. Considering the complexity of compost, it is extremely dif­ i I I ficult to attribute possible causes for this observation to any 2 3 4 6 2 3 4 6 one ingredient or class of ingredients in the compost treat­ Flush NO. Flush NO. ments. However, Maggioni et al. (11) reported that sub­ Figure 2. Flush study: Variations in agaritine concentrations of stitution of ammonium nitrogen in the compost of culti­ mushrooms harvested from six compost types at peak days offour vated mushrooms with urea resulted in reduced levels of selected flushes. I - Mean agaritine concentrations, dry weight most free amino acids in the harvested mushrooms. Con­ basis. II - Absolute amount of agaritine produced by mushrooms versely, elevated levels of aspartic acid, a.-alanine, valine harvested per average tray of compost (0.372 rrr). Bars with the and methionine were observed. Other workers (6) also same letter are not significantly different at the P 0.01 level. demonstrated that nutrient supplements, such as gelatin or hydrolyzed casein, added to compost also affected the nitol levels highest in mushrooms harvested from the peak composition of free amino acids in the harvested mus­ day of a given flush and directly correlated these levels hrooms. Agaritine, a derivative of , is also with crop yield. Additionally, Hammond and Nichols (2) classified as a free amino acid (8,9), Consequently, it is found mannitol levels lowest in mushrooms harvested be­ not surprising to observe significant variation in the tween flushes, whereas trehalose and glycogen were at agaritine content of mushrooms harvested from several their highest levels. Maggioni et al. (11) observed signifi­ compost types. However, actual isolation of the compost cant decreases in lysine, ornithine, aspartic acid and constituent(s) affecting the agaritine levels will require ad­ glutamic acid in fourth flush mushrooms compared to in­ ditional research. itial-break mushrooms. At the same time, they also noted A typical pattern of the cyclical yield of mushrooms increases in phenylalanine, proline and total free amino (flushing phenomenon) is illustrated in Fig. I. As normally acid nitrogen. occurs, the later flushes produced smaller yields than the Data derived from the compost study can be used to earlier flushes. Evidence exists that mushroom composi­ demonstrate that agaritine levels varied considerably in tion varies significantly among flushes of the crop cycle, mushrooms harvested from the peak days of different Parrish et al. (12) and Hammond et al. (2) both found man- flushes. A very strong tendency for agaritine concentra-

JOURNAL OF FOOD PROTECTION, VOL. 46, JUNE 1983 AGARITINE CONTENT IN MUSHROOMS 509 tions of mushrooms to increase with the later flushes is evi­ (such as PSU-35l) would more likely produce higher dent (Fig. 2, I). This tendency is especially noticeable con­ agaritine levels than a commercial strain where the compe­ sidering that the mean values were determined from mus­ tition has been artifically reduced or eliminated. It is en­ hrooms grown on six differing composts. When the data couraging to note that the more common and commercially were expressed as the absolute amount of agaritine in mus­ important strains contained lower concentrations of hrooms produced per unit surface area of compost (one agaritine than PSU-351. 2 tray = 0.372 m ), agaritine levels still increased with the TABLE 4. Mycelium study: Agaritine concentrations (gIlOO g later flushes (Fig. 2, II) despite the concomitant significant dry weight) in five strains of mycelium (Agaricus bisporus) grown reduction in yield of mushrooms (Fig. 1). This seems to in­ in liquid culture. dicate that the metabolic interaction between compost and Agaritine concentration mushrooms is changing during the later stages of the crop­ Strain No. (gllOO g dry weight) ping cycle stimulating the mushrooms to produce more PSU-352 0.032a b agaritine per sporocarp. If the nature of this interaction was PSU-310 0.015

more clearly defined, it might be possible to limit the PSU-265 0.014b Downloaded from http://meridian.allenpress.com/jfp/article-pdf/46/6/506/1655910/0362-028x-46_6_506.pdf by guest on 28 September 2021 b amount of agaritine produced. PSU-324 0.015 PSU-351 0.019a TABLE 3. Strain study: Agaritine concentrations (gil 00 g dry aRepresents the average of three replications. weight) measured for eight selected strains of fresh mushrooms bRepresents the average of two replications. grown from a natural compost. Mean agaritine Table 4 lists the agaritine concentrations in the concentration" mycelium of several strains of Agaricus bisporus grown in Strain Phenotype (gllOO g a liquid culture system. This is the first report of identifica­ No. color dry weight) tion of agaritine in the mycelium. The data indicate the PSU-324 Light cream 0.217 A quantities are probably 10 to 25 times less than those found PSU-345 Light cream 0.213 A in the fruiting bodies of the same strain (Table 3). Re­ PSU-348 Off-white 0.279 A search is continuing in this laboratory to determine whether CS-1b Off-white 0.170 A a relationship exists between agaritine levels in the PSU-31O White 0.232 A mycelium and fruiting bodies. PSU-342 White 0.278 A PSU-344 Brown 0.188 A CONCLUSIONS PSU-351 Brown 0.511 B aStrains means represent the average of five observations during Agaritine levels of Agaricus bisporus were shown to be the growing cycle with two determinations per observation. affected by: (a) composition of compost; (b) different Means followed by the same letter are not significantly different flushes of the crop cycle; and (c) the spawn-strain or iso­ at the P = 0.01 level. late. It is premature to conclude that synthetic composts in bCommercial spawn strain. general will produce mushrooms with elevated agaritine levels. Mushroom compost is an extremely complex Results of the strain study (Table 3) indicated little vari­ biological matrix. Two compost piles with identical pre­ ation in agaritine levels among strains when mean values composting ingredients may produce very dissimilar com­ from the entire crop were compared. Only one strain (PSU- posts due to difference in temperature and/or microbial 351) had significantly higher (P=O.Ol) agaritine levels flora during fermentation. Thus, each compost should be than the remaining seven strains. Also, no apparent re­ considered unique whenever strain, flush or compost lationship existed between mushroom color and agaritine evaluations are performed. levels. PSU-35l is classified as a "wild" strain of Agaricus bisporus since it was recently isolated from na­ ACKNOWLEDGMENTS ture by one of the authors (L. Schisler). It contained about twice as much agaritine (dry weight basis) as the other Paper No. 6429 in the Journal Series of The Pennsylvania Agricultural Experiment Station. Partial support for this investigation was furnished by commercially grown strains. Whether this apparent differ­ a grant from the American Mushroom Institute. ence in agaritine levels is of physiological, ecological or REFERENCES toxicological importance is only speculative at this time. However, in unpublished work done in this laboratory (S. I. Beelman, R. B., F. J. McArdle, and G. K. Parrish. 1974. Variations W. King, C. W. Fergus and R. B. Beelman), agaritine in the protein content and canned product yield of four important processing strains of the cultivated mushroom. Mushroom Sci. (0.02% aqueous solution) was demonstrated to inhibit (in 9:333-340. vitro) the vegetative growth of certain fungi known to be 2. Hammond, J. B. W., and R. Nichols. 1979. Carbohydrate "weed-mold" competitors, including isolates of Spicellum metabolism in Agaricus bisporus: changes in the non-structural car­ roseum and Trichoderma viride found in mushroom com­ bohydrates during periodic fruiting (flushing). New Phyto\. 83:723- post. It is possible that agaritine functions (in vivo) as an 730. 3. Hayes, W. A. 1978. Biological nature. pp. 191-217. In S. T. Chang antimycotic agent, inhibiting or preventing the growth of and W. A. Hayes (eds.) The biology and cultivation of edible mus­ competitive fungi. If this is true, it is logical to expect that hrooms. Academic Press, New York. a strain of Agaricus bisporus recently isolated from nature 4. Hayes, W. A. 1978. Nutrition, substrates, and principles of disease con't.p.5/3 JOURNAL OF FOOD PROTECTION, VOL. 46, JUNE 1983