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Pichia anomala as a biocontrol agent during storage of high-moisture feed grain under airtight conditions
Postharvest Biology and Technology 15 (1999) 175 – 184
Pichia anomala as a biocontrol agent during storage of high-moisture feed grain under airtight conditions Stina Petersson a, Nils Jonsson b, Johan Schnu¨rer a,* a
Department of Microbiology, SLU, Swedish Uni6ersity of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden b Swedish Institute of Agricultural Engineering, Box 7033, S-750 07 Uppsala, Sweden Received 5 March 1998; accepted 28 October 1998
Abstract Pichia anomala is antagonistic against a range of spoilage molds in vitro as well as against Penicillium roqueforti in high-moisture wheat during malfunctioning airtight storage in laboratory experiments. The use of Pichia anomala to improve the postharvest control of Penicillium roqueforti during airtight storage of feed grain was evaluated in outdoor silos. Inoculated and control winter wheat (cultivar Kosack) in 160-kg portions were stored at a water activity of 0.93 for 12 months in silos that were opened twice a week. During the first 2 months, inoculated Pichia anomala increased to about 107 colony-forming units (CFU)/g, while naturally occurring Pichia anomala in the treatments without inoculated yeast increased from 104 to 106 CFU/g. During the same period, CO2 concentrations increased to almost 70% and stabilized at 50–60%. During the coldest period, O2 concentrations of B 1% could be detected between samplings, whereas during the rest of the storage detectable O2 levels were only found immediately after sampling. There were no clear differences in CO2 or O2 levels between treatments. The inoculated Penicillium roqueforti did not grow during the storage period, probably owing to high numbers of Pichia anomala in combination with the high CO2 and low O2 concentrations in the silos. In laboratory experiments, it was found that Pichia anomala survived long-term storage in airtight sealed test tubes better at 15°C than at − 20°C. The aerobic stability of moist wheat after 10 and 12 months of silo storage was clearly enhanced by an initial inoculation with Pichia anomala. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Biocontrol; Yeast; Grain; Spoilage; Storage
1. Introduction
* Correspondent author. Tel.: +46-18-67-10-00; fax: +4618-67-33-92; e-mail: [email protected].
Penicillium roqueforti is one of the most important spoilage fungi in airtight stored cereals since it can grow at low partial pressures of O2 (\
0925-5214/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 9 8 ) 0 0 0 8 1 - 7
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0.14%), high levels of CO2 and low temperatures and is a potential mycotoxin producer (Lacey, 1989; Ha¨ggblom, 1990; Ohmomo and Kitamoto, 1994). The antagonistic yeast Pichia anomala (formerly Hansenula anomala (Kurtzman, 1984)) can reduce growth of Penicillium roqueforti both in vitro and in high-moisture cereal grain in a testtube version of a malfunctioning airtight storage system (Bjo¨rnberg and Schnu¨rer, 1993; Petersson and Schnu¨rer, 1995, 1998). Pichia anomala has also been shown to reduce ochratoxin A accumulation in co-culture with Penicillium 6errucosum, and toxin production was inhibited at levels of Pichia anomala lower than those inhibiting growth (Petersson et al., 1998). In Sweden, cereal grain is commonly harvested at a water content of 20 – 22%, corresponding to a water activity of 0.9. Mold growth in cereal grain is often prevented through high-temperature drying, although this is costly and energy demanding in a temperate climate. On farms where cereals are grown using conventional cultivation methods, about 60% of the total quantity of fuel used in plant husbandry operations is consumed in connection with the high-temperature drying of grain (Pick et al., 1989). Airtight storage, which is an alternative method of storing feed grain, relies on perfect sealing in combination with the anaerobic conditions caused by respiration of the grain itself and the adhering microorganisms. Airtight storage only requires about 2% of the energy consumed in high-temperature drying (calculated from Pick et al., 1989). However, leakage during airtight storage can easily lead to a deterioration of the conditions required for preservation. Changes in internal silo temperature (mostly due to solar radiation) and atmospheric pressure, together with increasing gas volume as the silo empties, lead to air movements through the relief valves and shell perforations, or through the emptying auger tube. In practice, grain is commonly not taken out from the silos every day. Instead, larger batches are stored with full access to air for several days. Techniques able to prevent mold spoilage and mycotoxin production would also be advantageous during this short-term storage.
At present, yeasts appear to be promising biocontrol agents, providing alternatives to chemical fungicides in the postharvest storage of fruits and vegetables (Arras, 1996; Chand-Goyal and Spotts, 1996; Lima et al., 1997; Piano et al., 1997). This potential benefit can be attributed to several properties of yeasts: they do not produce mycotoxins or allergenic spores; the vast majority are not pathogenic to humans or other animals; and many yeast species can grow at low levels of water availability on very different substrates and at low partial pressures of O2. In 1995, Candida oleophila Montrocher was registered for postharvest biocontrol as Aspire™ biofungicide by the Environmental Protection Agency in the United States (El-Neshawy, 1997). The product efficiently inhibits Penicillium spp. and Botrytis spp. growth on citrus and pome fruits. To evaluate the practical potential of Pichia anomala as a mold biocontrol agent we constructed 0.2-m3 pilot scale silos for airtight storage of moist grain. High-moisture wheat with various inoculum concentrations of Pichia anomala and Penicillium roqueforti were stored in the silos and sampled twice a week. Concentrations of O2 and CO2 were monitored and the microbial flora of the grains was followed over 13 months of storage. The mold suppressing ability of Pichia anomala during aerobic storage of wheat removed from the silos was evaluated after 10 and 12 months of airtight storage. In addition, survival of Pichia anomala in high-moisture wheat was evaluated at different temperatures during 15 months of airtight storage in test tubes.
2. Materials and methods
2.1. Microorganisms 2.1.1. Fungal isolates Penicillium roqueforti Thom (J5), a gift from Dr P. Ha¨ggblom, and Pichia anomala (Hansen) Kurtzman (J121) were originally isolated from stored grain. The fungi are stored in the fungal collection of the Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden. Pichia anomala was identified with
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the ID32C test (Biomerieux, Marcy l’Etoile, France) and through morphological studies (Kreger-van Rij, 1984; Kurtzman, 1984). Also, naturally occurring yeasts in the airtight pilotscale storage were identified using ID32C and through morphological studies. Identification of Pichia anomala and naturally occurring yeasts was confirmed at the Centraalbureau voor Schimmelcultures, Delft, The Netherlands. The molds were identified according to Pitt (1991)and Samson et al. (1995). Fungal cultures were maintained on slants of malt extract agar (MEA, Oxoid, Basingstoke, UK) at 4°C.
2.1.2. Inocula preparation Yeast suspensions were prepared by inoculating 1-l aliquots of yeast extract malt extract sucrose broth (0.2 g yeast extract (BBL, Meyland, France), 1.5 g malt extract (Oxoid)), 1.0 g sucrose (BDH, Poole, England) in 1.0 l distilled water) in 3-l Erlenmeyer flasks with a loopful of cells from a culture stored on MEA at 4°C. After incubation on a rotary shaker (100 rpm) at 25°C for 48 h, yeast cells were counted using a haemocytometer. The spore suspension of Penicillium roqueforti was prepared by collecting spores from 5 to 7-day-old colonies (grown on MEA-slants in flasks at 25°C) in peptone water (2 g peptone per l distilled water) with 0.015% Tween 80 added to assist dispersal of conidia. The spore concentration was estimated using a haemocytometer.
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mixing to obtain an even spore distribution. Thick-walled glass tubes (27 ml) were filled with approximately 17-g portions of inoculated grain and sealed with a butyl rubber stopper. The tubes were incubated at − 20, 2, 10 and 15°C. After 1, 2, 4, 6, 8 and 15 months, three tubes from each temperature were opened and analysed as described below (see Section 2.3).
2.2.2. Pilot -scale experiments 2.2.2.1. Experimental equipment. The silos each contained 0.21 m3 grain corresponding to 160 kg. To equalize differences between internal and external air pressure, each silo was equipped with both a breather bag and a two-way pressure relief valve consisting of a U-tube filled with a nonfreezing glycol/water solution (Fig. 1). Except when wheat was removed through the unloading
2.2. In 6itro and large scale tests 2.2.1. Sur6i6al of Pichia anomala in wheat stored airtight in test tubes Non-sterile winter wheat, cv. Kosack, stored at a water content of 10% at 20°C was moistened with tap water to obtain a water activity of 0.95. To equilibrate the moisture content, the grain was stored for 3–5 days at 2°C with frequent mixing. Water activity (aw) was measured at 22 – 25°C with an Aqua Lab CX-2 (Decagon Devices, Pullman, WA, USA). The high-moisture wheat grain was inoculated with the Pichia anomala cell suspension to about 6× 105 CFU per gram. The yeast was applied by adding the suspension as drops onto the grain and
Fig. 1. Schematic representation of the constructed silos, each containing 0.21 m3 wheat corresponding to 160 kg. To equalize differences between the internal and external air pressures, each silo was equipped with both a rubber breather bag and a two-way pressure-relief valve ( 9 60 mm static water gauge) consisting of a U-tube filled with a non-freezing glycol water solution. Air passed through the U-tube after the breather bag had been filled with \14 l of air or when 7 l of air was drained from the bag.
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spouts, no leakage could occur through the relief valve until the pressure exceeded 960 mm static water gauge. This pressure differential could not build up before the breather bag had either been filled with 14 l of air, or 7 l of air had been drained from it. Concentrations of CO2 and O2 in the silos were measured with an infrared monitor (accuracy9 0.5%) and a galvanic cell (accuracy91%) (GA 90, Geotechnical Instruments Limited, Leamington Spa, Warwickshire, UK), respectively. Pressure variations were continuously registered with a differential pressure meter (Model 600D, Innovex, Hopkins, MN, USA) in one of the silos. The temperature of the wheat was measured at each sampling time with an infrared thermometer (Model C-600, Linear Laboratories, Mountain View, CA, USA).
2.2.2.2. Pilot-scale inhibition experiments. Conventionally grown winter wheat (cv. Kosack) was harvested at a water content of 24% (aw 0.94) on 31 August 1996 and inoculated with the Penicillium roqueforti spore suspension to about 3×103 mold spores per g. The spores were applied by adding the suspension as drops onto the grain during mixing in a feed mixer to obtain an even spore distribution on the grain. The yeast was similarly inoculated to reach 0, 104 and 106 cells per g wheat and mixed with the wheat in that order. In the control treatment, which was mixed first to avoid cross contamination, only tap water was added. To get the same water activity, identical inoculum volumes were used in all experiments. The silos were filled with approximately 160 kg of inoculated wheat, using three replicates for each treatment. After sealing, the silos were placed in an outdoor shelter protected from rain and direct solar radiation for 13 months. The silos were initially sampled after 1 month of storage, whereafter they were sampled twice a week. The wheat samples, 1 kg each, were removed through the unloading spouts. Samples were taken after 5, 5.5, 6 and 8 weeks, and monthly thereafter. All samples (aliquots of about 20 g) were analyzed for molds, yeasts, aerobic bacteria and lactic acid bacteria as described in Section 2.3. Water activity in the wheat was mea-
sured on samples taken after 0, 8, 10 and 57 weeks. After 54 weeks of storage the silos (now containing 30–50 kg grain) were opened to obtain an elevated O2 concentration in the silos. First the caps of unloading spouts (diameter 44 mm) were removed, and after 3 days the connections to the breather bags (diameter 8 mm) were also removed. After 5 days, when the O2 concentration was about 18%, the silos were closed. Concentrations of CO2 and O2 were continuously monitored in one silo with wheat inoculated with 106 CFU of Pichia anomala per g until the O2 had been totally depleted. Thereafter the atmospheric composition was monitored in one silo containing uninoculated wheat. On two occasions, CO2 and O2 concentrations in all silos were monitored. The microbial flora was analyzed 1 week after closing the silos.
2.2.2.3. Aerobic stability after long term airtight storage of wheat. After 10 and 12 months of storage, 700 g grain from each silo was incubated for 3 weeks at 20°C in Erlenmeyer flasks plugged with cotton wool. Before sampling, the grain was thoroughly mixed. Growth of Penicillium roqueforti, Pichia anomala, aerobic bacteria and lactic acid bacteria were recorded frequently during the 3 weeks, as described below in Section 2.3. 2.3. Quantification of microbial growth Grain samples were diluted 10-fold with peptone water, soaked for 0.5 h and homogenized for 2 min at normal speed in a Stomacher 400 (Colworth, UK). To avoid growth inhibition by antagonistic yeast on agar plates intended for mold CFU counting, MEACC (MEA supplemented with 100 ppm chloramphenicol, C-0378 (Sigma, St. Louis, MO, USA, and 10 ppm cycloheximide, C-7698 Sigma, St. Louis, MO, USA) was used for surface plating 0.1 ml aliquots. Cycloheximide at 10 ppm inhibits the growth of Pichia anomala without affecting Penicillium roqueforti (Bjo¨rnberg and Schnu¨rer, 1993). Yeast growth was quantified on MEA supplemented with 100 ppm chloramphenicol (C-0378, Sigma, St. Louis, MO, USA; MEAC plates). Lactic acid bacteria and the total
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3.2. Pilot-scale inhibition experiments 3.2.1. En6ironmental conditions Variations in the temperature of the sampled wheat closely paralleled changes in the daily mean temperature of the outdoor air during the experiment (Fig. 3). Differences in air pressure between the gas in the silos and the external atmosphere were close to zero most of the time (data not shown). The pressure inside the silos was low enough to cause a movement of air into the silos through the pressure relief valve on only seven occasions during the first 2 months and on one occasion during weeks 28, 29, 44 and 46, respectively (data not shown). The water activity of the high-moisture wheat grain remained between 0.93 and 0.94 during the storage period (data not shown). Fig. 2. Survival of Pichia anomala inoculated in high-moisture winter wheat (aw 0.95) to 6 × 105 CFU/g and stored in airtight test tubes at −20 (), 2 ( ), 10 (), or 15°C () during 15 months. Data represent mean values 9 S.D. (n= 3).
number of aerobic bacteria were counted on MRS (Oxoid, Basingstoke, UK) (anaerobic incubation) and plate count agar (Oxoid, Basingstoke, UK), respectively, both supplemented with 0.1% Delvocid® (Gist-brocades, Delft, The Netherlands). Delvocid is a broad-spectrum fungicide containing natamycin as active agent.
3.2.2. Atmospheric conditions in the silos O2 levels inside the silos had already dropped below the detection limit (1%) 5 h after sealing (data not shown). The concentration of CO2 increased rapidly during the first week of the experiment (Fig. 4). After 2 months the concentration of CO2 had increased to its maximum, about 70%, in all silos. Once the temperature decreased below
3. Results
3.1. Effect of temperature on sur6i6al of Pichia anomala in airtight stored wheat The highest survival was obtained at 15°C where 1 ×106 CFU/g was detected after 15 months, corresponding to 160% of the inoculated level (Fig. 2). High numbers were also recovered at 10 and 2°C after 15 months, i.e. 2 ×105 and 1× 105 CFU/g, corresponding to 32 and 16% of the inoculated level respectively. Survival was reduced at − 20°C where 4×104 CFU/g were found after 8 months and 4 ×103 CFU/g after 15 months, corresponding to 6 and 0.6% of the starting value.
Fig. 3. Temperatures during the experimental period. The line represents daily mean air temperature and (") indicates the wheat temperature on the sampling occasions.
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Fig. 4. Carbon dioxide concentrations in silos with: non-inoculated wheat (), 103 CFU/g Penicillium roqueforti ( ), 103 CFU/g Penicillium roqueforti and 104 CFU/g Pichia anomala (), or 103 CFU/g Penicillium roqueforti and 106 CFU/g Pichia anomala (). Data represent mean values (n= 3). The coefficients of variation increased with increasing airspace in the silos to maximum values of 19, 11, 10 and 26%, respectively.
the freezing point after week 12, the average concentration of CO2 decreased and stabilized at between 50 and 60%. The CO2 levels in the silos diverged among the three replicates with increasing empty space in the silos. Thus, the coefficients of variation (CV) increased to between 10 and 20% after 30 weeks of storage, except in grain inoculated with 106 CFU/g Pichia anomala where a CV of between 20 and 26% was observed during the same time. During the period with temperatures below the freezing point, O2 concentrations in the silos fluctuated around the detection limit ( B1%), whereas during the rest of the period, O2 was only detected immediately after sampling.
3.2.3. Microbial population dynamics in airtight stored wheat in silos Inoculation with Pichia anomala aimed at reaching 104 and 106 CFU/g resulted in detected levels of 4× 104 and 5× 105 CFU/g, respectively. In addition, the wheat had a natural flora consisting of 1×104 CFU/g yeast. This yeast was identified as naturally occurring Pichia anomala on the
basis of morphology (e.g., hat-shaped ascospores) and growth physiology tests. After the first 5 weeks of storage in closed silos, the yeast numbers had increased to 6 × 104 CFU/g in the non-inoculated grain, to 2× 104 CFU/g in grain inoculated with Penicillium roqueforti, to 4×105 CFU/g when inoculated with 1×104 CFU of Pichia anomala/g, and to 9 ×105 CFU/g when inoculated with 5 × 105 CFU of Pichia anomala/g (Fig. 5a). After 8 weeks of storage, the amount of Pichia anomala in the two inoculated treatments was 4× 106 CFU/g, while the treatments not inoculated with yeast contained 4× 105 CFU/g grain. After 10 weeks of storage, the inoculated Pichia anomala had increased to 7×106 CFU/g in both treatments. Naturally occurring Pichia anomala had increased to about 6 ×105 CFU/g in the non-inoculated wheat. This difference persisted during weeks 20–35, i.e. from January to the end of April. During May with increasing temperatures, the yeast in the non-inoculated grain reached the level detected in the Pichia anomala inoculated grain, i.e. close to 107 CFU/g. Penicillium roqueforti was inoculated to 3×103 CFU/g in order to represent a high contamination level. However, this level of Penicillium roqueforti was recovered on only one occasion during the year (Fig. 5b). Levels were reduced to the detection limit, i.e. 102 CFU/g, during the initial 5 weeks when the silos were not opened. During the cold period, between weeks 10 and 35, CFU values increased slightly to between 102 and 103/g. At the end of the storage period the value for Penicillium roqueforti was back to 102 CFU/g again. In grain that had not been inoculated with Pichia anomala or Penicillium roqueforti, endogenous Penicillium roqueforti was only found on one of 16 sampling occasions, i.e. after 36 weeks of storage. Generally, no significant differences in Penicillium roqueforti CFU were found between the treatments (Fig. 5b). At the start of the experiment, about 102 CFU/ g of lactic acid bacteria were found in the inoculated wheat (data not shown). During storage, on rare occasions, B 103 CFU/g lactic acid bacteria was detected in the inoculated wheat. In the noninoculated silos, between 102 and 103 CFU/g lactic acid bacteria were found only until week 39 of
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Fig. 5. Growth of (a) Pichia anomala, (b) Penicillium roqueforti and (c) aerobic bacteria in high-moisture winter wheat (cv. Kosack) that was either: non-inoculated (), inoculated with 103 CFU/g Penicillium roqueforti ( ), 103 CFU/g Penicillium roqueforti and 104 CFU/g Pichia anomala (), or 103 CFU/g Penicillium roqueforti and 106 CFU/g Pichia anomala () and incubated in airtight silos. The least significant difference (L.S.D.) between mean values (n = 3, P =0.05) is in (a) 0.48, (b) 0.55 and (c) 0.48.
storage; thereafter, no lactic acid bacteria were detected in the non-inoculated wheat. Initially, the wheat contained 5×106 CFU/g of aerobic bacteria (Fig. 5c). During the 5 weeks of closed storage the amount decreased to 103 and 104 CFU/g, but levels recovered to varying degrees, reaching 105 CFU/g in the non-inoculated
treatment, 106 CFU/g in the treatment with Penicillium roqueforti only, and 104 CFU/g in both treatments with inoculated Pichia anomala plus Penicillium roqueforti. During weeks 20–50 (January to end of August) the level of aerobic bacteria remained more or less stable, with the exception of a small decrease in week 35–42 (May—June).
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During the simulated air leakage after 54 weeks of storage, the O2 concentration increased in all silos to ca 18% in 5 days (data not shown). Once the silo with wheat inoculated with 106 CFU Pichia anomala per g was closed, all detectable O2 was consumed within 20 h, whereas it took 46 h in the non-inoculated grain. One week after the planned air-leakage no increase in Penicillium roqueforti CFU could be detected for any of the treatments.
3.3. Aerobic stability of Pichia anomala inoculated wheat after long-term airtight storage In wheat stored under airtight conditions for 10 months and then exposed to air, Penicillium roqueforti inoculated alone reached 105 CFU/g after 6 days, whereas it took 12 days to reach the same level in wheat inoculated with 104 CFU/g and 14 days in wheat inoculated with 106 CFU/g Pichia anomala (Fig. 6). Similiar results were obtained with wheat after 12 months of airtight storage.
Fig. 6. Growth of Penicillium roqueforti in wheat removed from the silos and exposed to air after 10 months storage. Wheat that was non-inoculated (), inoculated with 103 CFU/ g Penicillium roqueforti ( ), 103 CFU/g Penicillium roqueforti and 104 CFU/g Pichia anomala (), or 103 CFU/g Penicillium roqueforti and 106 CFU/g Pichia anomala () was stored with full air supply at 20°C. Data represent mean values 9S.D. (n= 3).
4. Discussion Growth of Penicillium roqueforti, measured as colony-forming units (CFU), in the high-moisture wheat was almost totally inhibited during airtight storage in the silos. High CO2 and low O2 levels in the silos, as well as the high levels of inoculated and naturally occurring Pichia anomala in the wheat grain, contributed to the growth suppression of Penicillium roqueforti. When stored wheat was removed from the silos and exposed to air, the growth of Penicillium roqueforti was delayed by inoculated Pichia anomala in a dose-dependent manner. In test tube experiments with high-moisture wheat, Penicillium roqueforti growth was restricted by Pichia anomala inoculated to 103 CFU/ gram and almost totally inhibited by 104 CFU/g during 14 days of air leakage (Petersson and Schnu¨rer, 1995). In comparison, other antagonistic yeasts often are used at very high concentrations, e.g. 106 CFU/g or higher (Paster et al., 1993; Lurie et al., 1995; Schisler et al., 1995; Piano et al., 1997). However, the final amount of yeasts on fruits or vegetables is seldom reported. The constructed silos were intended to mimic the function of full scale airtight silos. However, the impenetrability to air might have been higher in these experimental silos. When wheat with a moisture content of 23% was stored in a completely sealed container, levels of CO2 as high as 95% were measured in the intergranular space (Hyde and Burell, 1982). In practice, because of imperfect sealing, etc., the concentration of CO2 often increases to only 15–25% (Harvey, 1965; Burmeister et al., 1966). In our study, using wheat at a moisture content of 24%, the level of CO2 in the silos stabilized at 50–60%, indicating some air exchange. The silos were allowed to remain open for 1 min twice a week. Rather than leading to a large amount of gas exchange, these events probably resulted in an equalization in pressure with the outside atmosphere. The system seemed to be robust with regard to mold-inhibiting ability even during periods of minor air leakage. Not even the intentional inflow of O2 and outflow of CO2 at the end of the storage period induced any growth of Penicillium roqueforti.
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Earlier investigations of Pichia anomala as a biocontrol agent have only been performed using remoistened grain (Petersson and Schnu¨rer, 1995, 1998; Petersson et al., 1998). Although physiological differences between freshly harvested and remoistened grain are likely to exist (Wilson and Jay, 1975) the biocontrol ability of Pichia anomala was also evident in freshly harvested wheat grain. Pichia anomala survived airtight storage in wheat filled test tubes for 15 months at temperatures ranging from − 20 to 15°C. Surprisingly, the survival of Pichia anomala was higher at 15°C than at − 20°C. At −20°C, less than 1% of the inoculated Pichia anomala was recovered after 15 months. When using Pichia anomala as a biocontrol agent in outdoor-stored cereals in a temperate climate, the yeast’s survival capacity at low temperature is important. However, the mean temperature in Sweden rarely remains below − 20°C for periods of more than a month or two. Yeast survival after 1 and 2 months at − 20°C were 56 and 48%, respectively, i.e. still at levels sufficient for biocontrol. When exposed to air, wheat grain stored airtight for 10–12 months spoiled faster when not initially inoculated with Pichia anomala, although it contained a high level of naturally occurring Pichia anomala. Without showing any observable increase in CFU itself, inoculated Pichia anomala doubled the time it took for inoculated Penicillium roqueforti to increase from 102 to 105 CFU/g. Thus, the antagonistic activity of Pichia anomala was not strictly associated with detectable growth, as has been suggested for Pichia guilliermondii (Paster et al., 1993). Counts of aerobic bacteria decreased with increasing initial inoculation levels of Pichia anomala. This reduction might have been due to competition for space and nutrients. Also, antibacterial compounds have been reported to be produced by yeast (McCormack et al., 1994). The stimulation of bacterial growth in wheat only inoculated with Penicillium roqueforti might have resulted from interactions between Penicillium roqueforti and bacteria. Lactic acid bacteria have been shown to stimulate the growth of
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Penicillium roqueforti (Hansen and Jakobsen, 1997). The Pichia anomala cells were applied by spraying a suspension onto the grain, followed by mixing in a feed blender to get an even distribution. During full-scale operations, in which hundreds of tons of grain are handled, it should be possible to add the yeast by inserting a spray jet in the transport screw when loading the silo. The subsequent mixing is likely to distribute the yeast cells evenly on the grain. When evaluating the technical potential of a new biocontrol system a careful hazard analysis is essential. Generally, Pichia anomala can grow at body temperature (Barnett et al., 1990), but human infections caused by Pichia anomala are rare, and only 13 cases of opportunistic infections had been reported in the English-language literature as of 1995 (Yamada et al., 1995). Further, co-cultivating Pichia anomala and Penicillium 6errucosum showed that ochratoxin A accumulation both in agar and wheat was even more sensitive to the presence of yeast than fungal growth (Petersson et al., 1998). Since Pichia anomala is a common member of the microflora of wheat (Lacey and Magan, 1991), the high level of Pichia anomala found in the freshly harvested winter wheat came as no surprise. Inoculation with Pichia anomala in cereal grain could thus be considered a way to improve the biocontrol effect of a microorganism already naturally occurring in cereals. In summary, our results indicate that the large-scale application of Pichia anomala to airtight stored high-moisture feed wheat could protect against postharvest spoilage. The yeast, in combination with low O2 and very high CO2 concentrations, inhibited growth of Penicillium roqueforti. Pichia anomala also quickly reduced O2 concentrations to levels below the detection threshold after air leakage to the system as well as improving post-storage aerobic stability even after long-term storage. To improve the biocontrol options, we are presently screening a variety of habitats in search of antagonistic yeast species that are active also at low water activities and low temperatures.
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Acknowledgements The technical assistance of Inger Ohlsson and Clas Jonsson is gratefully acknowledged. This investigation was financed by the Swedish Council for Forestry and Agricultural Research (SJFR), the Swedish Farmers Foundation for Agricultural Research (SLF), and the Foundation for Strategic Environmental Research (MISTRA).
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quality in drying and storage. Elsevier Science B.V, Amsterdam, pp. 77 – 118. Lima, G., Ippolito, A., Nigro, F., Salerno, M., 1997. Effectiveness of Auerobasidium pullulans and Candida oleophila against postharvest strawberry rots. Postharvest Biol. Technol. 10, 169 – 178. Lurie, S., Droby, S., Chalupowicz, L., Chalutz, E., 1995. Efficacy of Candida oleophila strain 182 in preventing Penicillium expansum infection of nectarine fruits. Phytoparasitica 23, 231 – 234. McCormack, P.J., Wildman, H.G., Jeffries, P., 1994. Production of antibacterial compounds by phylloplane-inhabiting yeast and yeast-like fungi. Appl. Environ. Microbiol. 60, 927 – 931. Ohmomo, S., Kitamoto, H.K., 1994. Detection of Roquefortines in Penicillium roqueforti isolated from moulded maize silage. J. Sci. Food Agric. 64, 211 – 215. Paster, N., Droby, S., Chalutz, E., Menasherov, M., Nitzan, R., Wilson, C.L., 1993. Evaluation of the potential of the yeast Pichia guilliermondii as a biocontrol agent against Aspergillus fla6us and fungi of stored soya beans. Mycol. Res. 10, 1201 – 1206. Petersson, S., Schnu¨rer, J., 1995. Biocontrol of mold in highmoisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cere6isiae. Appl. Environ. Microbiol. 61, 1027 – 1032. Petersson, S., Schnu¨rer, J., 1998. Pichia anomala as a biocontrol agent of Penicillium roqueforti in high-moisture wheat, rye, barley, and oats stored under airtight conditions. Can. J. Microbiol. 44, 471 – 476. Petersson, S., Wittrup Hansen, M., Axberg, K., Hult, K., Schnu¨rer, J., 1998. Ochratoxin A accumulation in cultures of Penicillium 6errucosum with the antagonistic yeast Pichia anomala and Saccharomyces cere6isiae. Mycol. Res. 102, 1003 – 1008. Piano, S., Neyrotti, V., Migheli, Q., Gullino, M.L., 1997. Biocontrol capability of Metschnikowia pulcherrima against Botrytis postharvest rot of apple. Postharvest Biol. Technol. 11, 131 – 140. Pick, E., Noren, O., Nielsen, V., 1989. Energy Consumption and Input – Output Relations of Field Operations. Food and Agriculture Organization of the United Nations, Rome. Pitt, J.I., 1991. A Laboratory Guide to Common Penicillium Species. CSIRO Food Research Laboratory, North Ryde, Australia. Samson, R.A., van Reenen-Hoekstra, E.S., Frisvad, J.C., Filtenborg, O., 1995. Introduction to Food-Borne Fungi. Centraalbureau voor Schimmelculture, Institute of the Royal Netherlands Academy of Arts and Sciences, Baarn. Schisler, D.A., Kurtzman, C.P., Bothast, R.J., Slininger, P.J., 1995. Evaluation of yeasts for biological control of Fusarium dry rot of potatoes. Am. Potato J. 72, 339 – 353. Wilson, D.M., Jay, E., 1975. Influence of modified atmosphere on aflatoxin production in high moisture corn. Appl. Environ. Microbiol. 29, 224 – 228. Yamada, S., Maruoka, T., Nagai, K., Tsumura, N., Yamada, T., Sakata, Y., Tominaga, K., Motohiro, T., kato, H., Makimura, K., Yamaguchi, H., 1995. Catheter-related infections by Hansenula anomala in children. Scand. J. Infect. Dis. 27, 85 – 87.
Pichia anomala as a biocontrol agent during storage of high-moisture feed grain under airtight conditions Stina Petersson a, Nils Jonsson b, Johan Schnu¨rer a,* a
Department of Microbiology, SLU, Swedish Uni6ersity of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden b Swedish Institute of Agricultural Engineering, Box 7033, S-750 07 Uppsala, Sweden Received 5 March 1998; accepted 28 October 1998
Abstract Pichia anomala is antagonistic against a range of spoilage molds in vitro as well as against Penicillium roqueforti in high-moisture wheat during malfunctioning airtight storage in laboratory experiments. The use of Pichia anomala to improve the postharvest control of Penicillium roqueforti during airtight storage of feed grain was evaluated in outdoor silos. Inoculated and control winter wheat (cultivar Kosack) in 160-kg portions were stored at a water activity of 0.93 for 12 months in silos that were opened twice a week. During the first 2 months, inoculated Pichia anomala increased to about 107 colony-forming units (CFU)/g, while naturally occurring Pichia anomala in the treatments without inoculated yeast increased from 104 to 106 CFU/g. During the same period, CO2 concentrations increased to almost 70% and stabilized at 50–60%. During the coldest period, O2 concentrations of B 1% could be detected between samplings, whereas during the rest of the storage detectable O2 levels were only found immediately after sampling. There were no clear differences in CO2 or O2 levels between treatments. The inoculated Penicillium roqueforti did not grow during the storage period, probably owing to high numbers of Pichia anomala in combination with the high CO2 and low O2 concentrations in the silos. In laboratory experiments, it was found that Pichia anomala survived long-term storage in airtight sealed test tubes better at 15°C than at − 20°C. The aerobic stability of moist wheat after 10 and 12 months of silo storage was clearly enhanced by an initial inoculation with Pichia anomala. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Biocontrol; Yeast; Grain; Spoilage; Storage
1. Introduction
* Correspondent author. Tel.: +46-18-67-10-00; fax: +4618-67-33-92; e-mail: [email protected].
Penicillium roqueforti is one of the most important spoilage fungi in airtight stored cereals since it can grow at low partial pressures of O2 (\
0925-5214/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 5 2 1 4 ( 9 8 ) 0 0 0 8 1 - 7
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0.14%), high levels of CO2 and low temperatures and is a potential mycotoxin producer (Lacey, 1989; Ha¨ggblom, 1990; Ohmomo and Kitamoto, 1994). The antagonistic yeast Pichia anomala (formerly Hansenula anomala (Kurtzman, 1984)) can reduce growth of Penicillium roqueforti both in vitro and in high-moisture cereal grain in a testtube version of a malfunctioning airtight storage system (Bjo¨rnberg and Schnu¨rer, 1993; Petersson and Schnu¨rer, 1995, 1998). Pichia anomala has also been shown to reduce ochratoxin A accumulation in co-culture with Penicillium 6errucosum, and toxin production was inhibited at levels of Pichia anomala lower than those inhibiting growth (Petersson et al., 1998). In Sweden, cereal grain is commonly harvested at a water content of 20 – 22%, corresponding to a water activity of 0.9. Mold growth in cereal grain is often prevented through high-temperature drying, although this is costly and energy demanding in a temperate climate. On farms where cereals are grown using conventional cultivation methods, about 60% of the total quantity of fuel used in plant husbandry operations is consumed in connection with the high-temperature drying of grain (Pick et al., 1989). Airtight storage, which is an alternative method of storing feed grain, relies on perfect sealing in combination with the anaerobic conditions caused by respiration of the grain itself and the adhering microorganisms. Airtight storage only requires about 2% of the energy consumed in high-temperature drying (calculated from Pick et al., 1989). However, leakage during airtight storage can easily lead to a deterioration of the conditions required for preservation. Changes in internal silo temperature (mostly due to solar radiation) and atmospheric pressure, together with increasing gas volume as the silo empties, lead to air movements through the relief valves and shell perforations, or through the emptying auger tube. In practice, grain is commonly not taken out from the silos every day. Instead, larger batches are stored with full access to air for several days. Techniques able to prevent mold spoilage and mycotoxin production would also be advantageous during this short-term storage.
At present, yeasts appear to be promising biocontrol agents, providing alternatives to chemical fungicides in the postharvest storage of fruits and vegetables (Arras, 1996; Chand-Goyal and Spotts, 1996; Lima et al., 1997; Piano et al., 1997). This potential benefit can be attributed to several properties of yeasts: they do not produce mycotoxins or allergenic spores; the vast majority are not pathogenic to humans or other animals; and many yeast species can grow at low levels of water availability on very different substrates and at low partial pressures of O2. In 1995, Candida oleophila Montrocher was registered for postharvest biocontrol as Aspire™ biofungicide by the Environmental Protection Agency in the United States (El-Neshawy, 1997). The product efficiently inhibits Penicillium spp. and Botrytis spp. growth on citrus and pome fruits. To evaluate the practical potential of Pichia anomala as a mold biocontrol agent we constructed 0.2-m3 pilot scale silos for airtight storage of moist grain. High-moisture wheat with various inoculum concentrations of Pichia anomala and Penicillium roqueforti were stored in the silos and sampled twice a week. Concentrations of O2 and CO2 were monitored and the microbial flora of the grains was followed over 13 months of storage. The mold suppressing ability of Pichia anomala during aerobic storage of wheat removed from the silos was evaluated after 10 and 12 months of airtight storage. In addition, survival of Pichia anomala in high-moisture wheat was evaluated at different temperatures during 15 months of airtight storage in test tubes.
2. Materials and methods
2.1. Microorganisms 2.1.1. Fungal isolates Penicillium roqueforti Thom (J5), a gift from Dr P. Ha¨ggblom, and Pichia anomala (Hansen) Kurtzman (J121) were originally isolated from stored grain. The fungi are stored in the fungal collection of the Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden. Pichia anomala was identified with
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the ID32C test (Biomerieux, Marcy l’Etoile, France) and through morphological studies (Kreger-van Rij, 1984; Kurtzman, 1984). Also, naturally occurring yeasts in the airtight pilotscale storage were identified using ID32C and through morphological studies. Identification of Pichia anomala and naturally occurring yeasts was confirmed at the Centraalbureau voor Schimmelcultures, Delft, The Netherlands. The molds were identified according to Pitt (1991)and Samson et al. (1995). Fungal cultures were maintained on slants of malt extract agar (MEA, Oxoid, Basingstoke, UK) at 4°C.
2.1.2. Inocula preparation Yeast suspensions were prepared by inoculating 1-l aliquots of yeast extract malt extract sucrose broth (0.2 g yeast extract (BBL, Meyland, France), 1.5 g malt extract (Oxoid)), 1.0 g sucrose (BDH, Poole, England) in 1.0 l distilled water) in 3-l Erlenmeyer flasks with a loopful of cells from a culture stored on MEA at 4°C. After incubation on a rotary shaker (100 rpm) at 25°C for 48 h, yeast cells were counted using a haemocytometer. The spore suspension of Penicillium roqueforti was prepared by collecting spores from 5 to 7-day-old colonies (grown on MEA-slants in flasks at 25°C) in peptone water (2 g peptone per l distilled water) with 0.015% Tween 80 added to assist dispersal of conidia. The spore concentration was estimated using a haemocytometer.
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mixing to obtain an even spore distribution. Thick-walled glass tubes (27 ml) were filled with approximately 17-g portions of inoculated grain and sealed with a butyl rubber stopper. The tubes were incubated at − 20, 2, 10 and 15°C. After 1, 2, 4, 6, 8 and 15 months, three tubes from each temperature were opened and analysed as described below (see Section 2.3).
2.2.2. Pilot -scale experiments 2.2.2.1. Experimental equipment. The silos each contained 0.21 m3 grain corresponding to 160 kg. To equalize differences between internal and external air pressure, each silo was equipped with both a breather bag and a two-way pressure relief valve consisting of a U-tube filled with a nonfreezing glycol/water solution (Fig. 1). Except when wheat was removed through the unloading
2.2. In 6itro and large scale tests 2.2.1. Sur6i6al of Pichia anomala in wheat stored airtight in test tubes Non-sterile winter wheat, cv. Kosack, stored at a water content of 10% at 20°C was moistened with tap water to obtain a water activity of 0.95. To equilibrate the moisture content, the grain was stored for 3–5 days at 2°C with frequent mixing. Water activity (aw) was measured at 22 – 25°C with an Aqua Lab CX-2 (Decagon Devices, Pullman, WA, USA). The high-moisture wheat grain was inoculated with the Pichia anomala cell suspension to about 6× 105 CFU per gram. The yeast was applied by adding the suspension as drops onto the grain and
Fig. 1. Schematic representation of the constructed silos, each containing 0.21 m3 wheat corresponding to 160 kg. To equalize differences between the internal and external air pressures, each silo was equipped with both a rubber breather bag and a two-way pressure-relief valve ( 9 60 mm static water gauge) consisting of a U-tube filled with a non-freezing glycol water solution. Air passed through the U-tube after the breather bag had been filled with \14 l of air or when 7 l of air was drained from the bag.
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spouts, no leakage could occur through the relief valve until the pressure exceeded 960 mm static water gauge. This pressure differential could not build up before the breather bag had either been filled with 14 l of air, or 7 l of air had been drained from it. Concentrations of CO2 and O2 in the silos were measured with an infrared monitor (accuracy9 0.5%) and a galvanic cell (accuracy91%) (GA 90, Geotechnical Instruments Limited, Leamington Spa, Warwickshire, UK), respectively. Pressure variations were continuously registered with a differential pressure meter (Model 600D, Innovex, Hopkins, MN, USA) in one of the silos. The temperature of the wheat was measured at each sampling time with an infrared thermometer (Model C-600, Linear Laboratories, Mountain View, CA, USA).
2.2.2.2. Pilot-scale inhibition experiments. Conventionally grown winter wheat (cv. Kosack) was harvested at a water content of 24% (aw 0.94) on 31 August 1996 and inoculated with the Penicillium roqueforti spore suspension to about 3×103 mold spores per g. The spores were applied by adding the suspension as drops onto the grain during mixing in a feed mixer to obtain an even spore distribution on the grain. The yeast was similarly inoculated to reach 0, 104 and 106 cells per g wheat and mixed with the wheat in that order. In the control treatment, which was mixed first to avoid cross contamination, only tap water was added. To get the same water activity, identical inoculum volumes were used in all experiments. The silos were filled with approximately 160 kg of inoculated wheat, using three replicates for each treatment. After sealing, the silos were placed in an outdoor shelter protected from rain and direct solar radiation for 13 months. The silos were initially sampled after 1 month of storage, whereafter they were sampled twice a week. The wheat samples, 1 kg each, were removed through the unloading spouts. Samples were taken after 5, 5.5, 6 and 8 weeks, and monthly thereafter. All samples (aliquots of about 20 g) were analyzed for molds, yeasts, aerobic bacteria and lactic acid bacteria as described in Section 2.3. Water activity in the wheat was mea-
sured on samples taken after 0, 8, 10 and 57 weeks. After 54 weeks of storage the silos (now containing 30–50 kg grain) were opened to obtain an elevated O2 concentration in the silos. First the caps of unloading spouts (diameter 44 mm) were removed, and after 3 days the connections to the breather bags (diameter 8 mm) were also removed. After 5 days, when the O2 concentration was about 18%, the silos were closed. Concentrations of CO2 and O2 were continuously monitored in one silo with wheat inoculated with 106 CFU of Pichia anomala per g until the O2 had been totally depleted. Thereafter the atmospheric composition was monitored in one silo containing uninoculated wheat. On two occasions, CO2 and O2 concentrations in all silos were monitored. The microbial flora was analyzed 1 week after closing the silos.
2.2.2.3. Aerobic stability after long term airtight storage of wheat. After 10 and 12 months of storage, 700 g grain from each silo was incubated for 3 weeks at 20°C in Erlenmeyer flasks plugged with cotton wool. Before sampling, the grain was thoroughly mixed. Growth of Penicillium roqueforti, Pichia anomala, aerobic bacteria and lactic acid bacteria were recorded frequently during the 3 weeks, as described below in Section 2.3. 2.3. Quantification of microbial growth Grain samples were diluted 10-fold with peptone water, soaked for 0.5 h and homogenized for 2 min at normal speed in a Stomacher 400 (Colworth, UK). To avoid growth inhibition by antagonistic yeast on agar plates intended for mold CFU counting, MEACC (MEA supplemented with 100 ppm chloramphenicol, C-0378 (Sigma, St. Louis, MO, USA, and 10 ppm cycloheximide, C-7698 Sigma, St. Louis, MO, USA) was used for surface plating 0.1 ml aliquots. Cycloheximide at 10 ppm inhibits the growth of Pichia anomala without affecting Penicillium roqueforti (Bjo¨rnberg and Schnu¨rer, 1993). Yeast growth was quantified on MEA supplemented with 100 ppm chloramphenicol (C-0378, Sigma, St. Louis, MO, USA; MEAC plates). Lactic acid bacteria and the total
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3.2. Pilot-scale inhibition experiments 3.2.1. En6ironmental conditions Variations in the temperature of the sampled wheat closely paralleled changes in the daily mean temperature of the outdoor air during the experiment (Fig. 3). Differences in air pressure between the gas in the silos and the external atmosphere were close to zero most of the time (data not shown). The pressure inside the silos was low enough to cause a movement of air into the silos through the pressure relief valve on only seven occasions during the first 2 months and on one occasion during weeks 28, 29, 44 and 46, respectively (data not shown). The water activity of the high-moisture wheat grain remained between 0.93 and 0.94 during the storage period (data not shown). Fig. 2. Survival of Pichia anomala inoculated in high-moisture winter wheat (aw 0.95) to 6 × 105 CFU/g and stored in airtight test tubes at −20 (), 2 ( ), 10 (), or 15°C () during 15 months. Data represent mean values 9 S.D. (n= 3).
number of aerobic bacteria were counted on MRS (Oxoid, Basingstoke, UK) (anaerobic incubation) and plate count agar (Oxoid, Basingstoke, UK), respectively, both supplemented with 0.1% Delvocid® (Gist-brocades, Delft, The Netherlands). Delvocid is a broad-spectrum fungicide containing natamycin as active agent.
3.2.2. Atmospheric conditions in the silos O2 levels inside the silos had already dropped below the detection limit (1%) 5 h after sealing (data not shown). The concentration of CO2 increased rapidly during the first week of the experiment (Fig. 4). After 2 months the concentration of CO2 had increased to its maximum, about 70%, in all silos. Once the temperature decreased below
3. Results
3.1. Effect of temperature on sur6i6al of Pichia anomala in airtight stored wheat The highest survival was obtained at 15°C where 1 ×106 CFU/g was detected after 15 months, corresponding to 160% of the inoculated level (Fig. 2). High numbers were also recovered at 10 and 2°C after 15 months, i.e. 2 ×105 and 1× 105 CFU/g, corresponding to 32 and 16% of the inoculated level respectively. Survival was reduced at − 20°C where 4×104 CFU/g were found after 8 months and 4 ×103 CFU/g after 15 months, corresponding to 6 and 0.6% of the starting value.
Fig. 3. Temperatures during the experimental period. The line represents daily mean air temperature and (") indicates the wheat temperature on the sampling occasions.
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Fig. 4. Carbon dioxide concentrations in silos with: non-inoculated wheat (), 103 CFU/g Penicillium roqueforti ( ), 103 CFU/g Penicillium roqueforti and 104 CFU/g Pichia anomala (), or 103 CFU/g Penicillium roqueforti and 106 CFU/g Pichia anomala (). Data represent mean values (n= 3). The coefficients of variation increased with increasing airspace in the silos to maximum values of 19, 11, 10 and 26%, respectively.
the freezing point after week 12, the average concentration of CO2 decreased and stabilized at between 50 and 60%. The CO2 levels in the silos diverged among the three replicates with increasing empty space in the silos. Thus, the coefficients of variation (CV) increased to between 10 and 20% after 30 weeks of storage, except in grain inoculated with 106 CFU/g Pichia anomala where a CV of between 20 and 26% was observed during the same time. During the period with temperatures below the freezing point, O2 concentrations in the silos fluctuated around the detection limit ( B1%), whereas during the rest of the period, O2 was only detected immediately after sampling.
3.2.3. Microbial population dynamics in airtight stored wheat in silos Inoculation with Pichia anomala aimed at reaching 104 and 106 CFU/g resulted in detected levels of 4× 104 and 5× 105 CFU/g, respectively. In addition, the wheat had a natural flora consisting of 1×104 CFU/g yeast. This yeast was identified as naturally occurring Pichia anomala on the
basis of morphology (e.g., hat-shaped ascospores) and growth physiology tests. After the first 5 weeks of storage in closed silos, the yeast numbers had increased to 6 × 104 CFU/g in the non-inoculated grain, to 2× 104 CFU/g in grain inoculated with Penicillium roqueforti, to 4×105 CFU/g when inoculated with 1×104 CFU of Pichia anomala/g, and to 9 ×105 CFU/g when inoculated with 5 × 105 CFU of Pichia anomala/g (Fig. 5a). After 8 weeks of storage, the amount of Pichia anomala in the two inoculated treatments was 4× 106 CFU/g, while the treatments not inoculated with yeast contained 4× 105 CFU/g grain. After 10 weeks of storage, the inoculated Pichia anomala had increased to 7×106 CFU/g in both treatments. Naturally occurring Pichia anomala had increased to about 6 ×105 CFU/g in the non-inoculated wheat. This difference persisted during weeks 20–35, i.e. from January to the end of April. During May with increasing temperatures, the yeast in the non-inoculated grain reached the level detected in the Pichia anomala inoculated grain, i.e. close to 107 CFU/g. Penicillium roqueforti was inoculated to 3×103 CFU/g in order to represent a high contamination level. However, this level of Penicillium roqueforti was recovered on only one occasion during the year (Fig. 5b). Levels were reduced to the detection limit, i.e. 102 CFU/g, during the initial 5 weeks when the silos were not opened. During the cold period, between weeks 10 and 35, CFU values increased slightly to between 102 and 103/g. At the end of the storage period the value for Penicillium roqueforti was back to 102 CFU/g again. In grain that had not been inoculated with Pichia anomala or Penicillium roqueforti, endogenous Penicillium roqueforti was only found on one of 16 sampling occasions, i.e. after 36 weeks of storage. Generally, no significant differences in Penicillium roqueforti CFU were found between the treatments (Fig. 5b). At the start of the experiment, about 102 CFU/ g of lactic acid bacteria were found in the inoculated wheat (data not shown). During storage, on rare occasions, B 103 CFU/g lactic acid bacteria was detected in the inoculated wheat. In the noninoculated silos, between 102 and 103 CFU/g lactic acid bacteria were found only until week 39 of
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Fig. 5. Growth of (a) Pichia anomala, (b) Penicillium roqueforti and (c) aerobic bacteria in high-moisture winter wheat (cv. Kosack) that was either: non-inoculated (), inoculated with 103 CFU/g Penicillium roqueforti ( ), 103 CFU/g Penicillium roqueforti and 104 CFU/g Pichia anomala (), or 103 CFU/g Penicillium roqueforti and 106 CFU/g Pichia anomala () and incubated in airtight silos. The least significant difference (L.S.D.) between mean values (n = 3, P =0.05) is in (a) 0.48, (b) 0.55 and (c) 0.48.
storage; thereafter, no lactic acid bacteria were detected in the non-inoculated wheat. Initially, the wheat contained 5×106 CFU/g of aerobic bacteria (Fig. 5c). During the 5 weeks of closed storage the amount decreased to 103 and 104 CFU/g, but levels recovered to varying degrees, reaching 105 CFU/g in the non-inoculated
treatment, 106 CFU/g in the treatment with Penicillium roqueforti only, and 104 CFU/g in both treatments with inoculated Pichia anomala plus Penicillium roqueforti. During weeks 20–50 (January to end of August) the level of aerobic bacteria remained more or less stable, with the exception of a small decrease in week 35–42 (May—June).
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During the simulated air leakage after 54 weeks of storage, the O2 concentration increased in all silos to ca 18% in 5 days (data not shown). Once the silo with wheat inoculated with 106 CFU Pichia anomala per g was closed, all detectable O2 was consumed within 20 h, whereas it took 46 h in the non-inoculated grain. One week after the planned air-leakage no increase in Penicillium roqueforti CFU could be detected for any of the treatments.
3.3. Aerobic stability of Pichia anomala inoculated wheat after long-term airtight storage In wheat stored under airtight conditions for 10 months and then exposed to air, Penicillium roqueforti inoculated alone reached 105 CFU/g after 6 days, whereas it took 12 days to reach the same level in wheat inoculated with 104 CFU/g and 14 days in wheat inoculated with 106 CFU/g Pichia anomala (Fig. 6). Similiar results were obtained with wheat after 12 months of airtight storage.
Fig. 6. Growth of Penicillium roqueforti in wheat removed from the silos and exposed to air after 10 months storage. Wheat that was non-inoculated (), inoculated with 103 CFU/ g Penicillium roqueforti ( ), 103 CFU/g Penicillium roqueforti and 104 CFU/g Pichia anomala (), or 103 CFU/g Penicillium roqueforti and 106 CFU/g Pichia anomala () was stored with full air supply at 20°C. Data represent mean values 9S.D. (n= 3).
4. Discussion Growth of Penicillium roqueforti, measured as colony-forming units (CFU), in the high-moisture wheat was almost totally inhibited during airtight storage in the silos. High CO2 and low O2 levels in the silos, as well as the high levels of inoculated and naturally occurring Pichia anomala in the wheat grain, contributed to the growth suppression of Penicillium roqueforti. When stored wheat was removed from the silos and exposed to air, the growth of Penicillium roqueforti was delayed by inoculated Pichia anomala in a dose-dependent manner. In test tube experiments with high-moisture wheat, Penicillium roqueforti growth was restricted by Pichia anomala inoculated to 103 CFU/ gram and almost totally inhibited by 104 CFU/g during 14 days of air leakage (Petersson and Schnu¨rer, 1995). In comparison, other antagonistic yeasts often are used at very high concentrations, e.g. 106 CFU/g or higher (Paster et al., 1993; Lurie et al., 1995; Schisler et al., 1995; Piano et al., 1997). However, the final amount of yeasts on fruits or vegetables is seldom reported. The constructed silos were intended to mimic the function of full scale airtight silos. However, the impenetrability to air might have been higher in these experimental silos. When wheat with a moisture content of 23% was stored in a completely sealed container, levels of CO2 as high as 95% were measured in the intergranular space (Hyde and Burell, 1982). In practice, because of imperfect sealing, etc., the concentration of CO2 often increases to only 15–25% (Harvey, 1965; Burmeister et al., 1966). In our study, using wheat at a moisture content of 24%, the level of CO2 in the silos stabilized at 50–60%, indicating some air exchange. The silos were allowed to remain open for 1 min twice a week. Rather than leading to a large amount of gas exchange, these events probably resulted in an equalization in pressure with the outside atmosphere. The system seemed to be robust with regard to mold-inhibiting ability even during periods of minor air leakage. Not even the intentional inflow of O2 and outflow of CO2 at the end of the storage period induced any growth of Penicillium roqueforti.
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Earlier investigations of Pichia anomala as a biocontrol agent have only been performed using remoistened grain (Petersson and Schnu¨rer, 1995, 1998; Petersson et al., 1998). Although physiological differences between freshly harvested and remoistened grain are likely to exist (Wilson and Jay, 1975) the biocontrol ability of Pichia anomala was also evident in freshly harvested wheat grain. Pichia anomala survived airtight storage in wheat filled test tubes for 15 months at temperatures ranging from − 20 to 15°C. Surprisingly, the survival of Pichia anomala was higher at 15°C than at − 20°C. At −20°C, less than 1% of the inoculated Pichia anomala was recovered after 15 months. When using Pichia anomala as a biocontrol agent in outdoor-stored cereals in a temperate climate, the yeast’s survival capacity at low temperature is important. However, the mean temperature in Sweden rarely remains below − 20°C for periods of more than a month or two. Yeast survival after 1 and 2 months at − 20°C were 56 and 48%, respectively, i.e. still at levels sufficient for biocontrol. When exposed to air, wheat grain stored airtight for 10–12 months spoiled faster when not initially inoculated with Pichia anomala, although it contained a high level of naturally occurring Pichia anomala. Without showing any observable increase in CFU itself, inoculated Pichia anomala doubled the time it took for inoculated Penicillium roqueforti to increase from 102 to 105 CFU/g. Thus, the antagonistic activity of Pichia anomala was not strictly associated with detectable growth, as has been suggested for Pichia guilliermondii (Paster et al., 1993). Counts of aerobic bacteria decreased with increasing initial inoculation levels of Pichia anomala. This reduction might have been due to competition for space and nutrients. Also, antibacterial compounds have been reported to be produced by yeast (McCormack et al., 1994). The stimulation of bacterial growth in wheat only inoculated with Penicillium roqueforti might have resulted from interactions between Penicillium roqueforti and bacteria. Lactic acid bacteria have been shown to stimulate the growth of
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Penicillium roqueforti (Hansen and Jakobsen, 1997). The Pichia anomala cells were applied by spraying a suspension onto the grain, followed by mixing in a feed blender to get an even distribution. During full-scale operations, in which hundreds of tons of grain are handled, it should be possible to add the yeast by inserting a spray jet in the transport screw when loading the silo. The subsequent mixing is likely to distribute the yeast cells evenly on the grain. When evaluating the technical potential of a new biocontrol system a careful hazard analysis is essential. Generally, Pichia anomala can grow at body temperature (Barnett et al., 1990), but human infections caused by Pichia anomala are rare, and only 13 cases of opportunistic infections had been reported in the English-language literature as of 1995 (Yamada et al., 1995). Further, co-cultivating Pichia anomala and Penicillium 6errucosum showed that ochratoxin A accumulation both in agar and wheat was even more sensitive to the presence of yeast than fungal growth (Petersson et al., 1998). Since Pichia anomala is a common member of the microflora of wheat (Lacey and Magan, 1991), the high level of Pichia anomala found in the freshly harvested winter wheat came as no surprise. Inoculation with Pichia anomala in cereal grain could thus be considered a way to improve the biocontrol effect of a microorganism already naturally occurring in cereals. In summary, our results indicate that the large-scale application of Pichia anomala to airtight stored high-moisture feed wheat could protect against postharvest spoilage. The yeast, in combination with low O2 and very high CO2 concentrations, inhibited growth of Penicillium roqueforti. Pichia anomala also quickly reduced O2 concentrations to levels below the detection threshold after air leakage to the system as well as improving post-storage aerobic stability even after long-term storage. To improve the biocontrol options, we are presently screening a variety of habitats in search of antagonistic yeast species that are active also at low water activities and low temperatures.
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Acknowledgements The technical assistance of Inger Ohlsson and Clas Jonsson is gratefully acknowledged. This investigation was financed by the Swedish Council for Forestry and Agricultural Research (SJFR), the Swedish Farmers Foundation for Agricultural Research (SLF), and the Foundation for Strategic Environmental Research (MISTRA).
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