The effect of adding water into the mixer on pelleting efficiency and pellet quality in diets for finishing pigs without and with use of an expander

Two diets for finishing pigs were used to determine the effect of adding water into the mixer on processing characteristics and pellet quality. Diet 1 was based on barley, oats and soybean meal (barley-based diet) and Diet 2 was based on maize and soybean meal (maize-based diet). Both diets were produced without and with use of an expander. Adding up to 120 g water/kg into the mixer prior to steam conditioning and pelleting (steam conditioning) of the barley-based diet, improved pelleting efficiency, pellet durability index (PDI) and modified PDI (with five hexagonal nuts added into the tumbling box), by 15, 10 and 10%, respectively. When water was added into the mixer prior to expanding the barley-based diet, PDI increased from 93 to 95% and modified PDI from 91 to 94%. The maize-based diet was processed with motor load held constant to maximize feed mill output, and production rate held constant to minimize energy use. At a constant motor load and a constant production rate, adding up to 30 g water/kg into the maize-based diet prior to steam conditioning increased pelleting efficiency by 22 and 9%, PDI was improved from 84 to 89% and from 79 to 87%, respectively. Expander conditioning of the maize-based diet increased PDI from 92% at none to 94% at 30 g/kg water addition. Correspondingly, modified PDI was increased from 89 to 93% at none and 30 g water/kg into the mixer prior to expander conditioning. The overall conclusion from the experiment was that adding water into the mixer before steam conditioning improved pelleting efficiency and pellet quality in barley- and maize-based diet for finishing pigs.     

Effect of pellet size and pellet quality on pig performance

An experiment involving 1360 growing finishing pigs was undertaken to examine the effect of pellet size and pellet quality, as measured by the Holmen pellet durability test, on pig performance. A barley and soya bean meal diet was used and pellets were of two sizes, 5 and 10 mm diameter. Pellet quality was varied to give two types of pellet by steam conditioning and screening procedure during the pelleting process. The mean difference obtained in pellet durability was 11%.

Pig performance between 30 and 80 kg liveweight was not affected by either pellet size or pellet quality. There were small non-significant trends in favour of both the smaller pellets and the lower quality pellets. These trends, of the order of 1% or less, followed the same pattern as the dry matter content of the diets. The smaller diameter pellets were dried more efficiently in the cooling process and the low durability pellets had less steam added during the manufacturing process, which was reflected in the dry matter content of the finished diets.     

The Role of Biomass Composition and Steam Treatment on Durability of Pellets

Steam treatment has been reported to improve the durability of wood pellet likely by changing the physical and chemical structures of wood particles, but published literature is inconclusive about which structure change is the major reason for enhanced durability. In this work, steam treatment was combined either with alkaline or with SO₂ to study. The solids obtained after steam treatments along with a control sample were dried and each was compacted into pellets. The pellets were then tested for durability. Steam treatment alone dominated improvements in durability. The pellet durability increased with the amount of xylose, but xylose performed better in the pellet from raw poplar than did in the pellet from treated poplar. Water-soluble components contributed a maximum 4% of the durability of poplar pellets. The addition of lignin and sugars to substrates after steam treatment did not improve durability significantly. The surface modification that took place as a result of size reduction during steam treatment was the major reason, contributing about 50% of the durability of the pellet from steam-treated poplar. The acidity of steam treatment slightly affected the relative contributions of these structure changes on the durability. The new knowledge helps tailor the chemical and/or mechanical pretreatment involved in pelleting biomass to durable pellets.     

High quality biofuel pellet production from pre-compacted low density raw materials

In this study, pre-compaction was evaluated as a method to enhance stable reed canary grass pellet production. An experimental design of the factors raw material moisture content, steam addition, raw material bulk density, and die temperature was used to find production conditions for high quality pellets by multiple linear regression modelling of responses. Response variables being modelled were variability of pelletizer current (as a measurement of uneven production), pellet bulk density, and pellet durability. By pre-compacting the raw material from a bulk density of 150 kg/m3 to 270kg/m3, continuous production could be obtained at minimum raw material moisture content of 13.8%. Bulk density and durability were both highly correlated to raw material moisture content, but showed different optima. Multiple response optimization was used to target process settings for production of high quality reed canary grass pellets with bulk density >650kg/m3 and durability >97.5%.     

Lab and Bench-Scale Pelletization of Torrefied Wood Chips—Process Optimization and Pellet Quality

Combined torrefaction and pelletization is used to increase the fuel value of biomass by increasing its energy density and improving its handling and combustion properties. In the present study, a single-pellet press tool was used to screen for the effects of pellet die temperature, moisture content, additive addition, and the degree of torrefaction on the pelletizing properties and pellet quality, i.e., density, static friction, and pellet strength. Results were compared with pellet production using a bench-scale pelletizer. The results indicate that friction is the key factor when scaling up from single-pellet press to bench-scale pelletizer. Tuning moisture content or increasing the die temperature did not ease the pellet production of torrefied wood chips significantly. The addition of rapeseed oil as a lubricant reduced the static friction by half and stabilized pellet production; however, the pellet quality, strength, and density were negatively affected. The pellets produced from pine wood torrefied at 250 and 280 °C were shorter than pellets produced from untreated wood and their quality did not match conventional wood pellet standards. However, the heating value of the torrefied pellets was higher and the particle size distribution after grinding the pellets was more uniform compared to conventional wood pellets.     

Activity of supplemental enzymes and their effect on nutrient utilization and growth performance of growing chickens as affected by pelleting temperature

Activity of supplemental enzymes in a barley‐soybean‐maize based diet at 60, 75 and 90°C pelleting temperatures was studied using feed viscosity, in‐vitro enzyme activity and broiler performance data.

High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75°C, but reduced by 73% in feed processed at 90°C. Enzymes increased the weight gain of broilers by 11.1% at 90°C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75°C. Reduced feed intake and daily weight gain observed at 90° C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes.

It is concluded that pelleting temperatures as high as 90°C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.     

Shelf-life of a Biocontrol Pseudomonas putida Applied to Sugar Beet Seeds Using Commercial Coatings

Pseudomonas putida 40RNF is a putative biological control agent (BCA) of Pythium damping-off of sugar beet. The survival of 40RNF during commercial seed treatment and its subsequent shelf-life (i.e. long-term viability and biocontrol activity) were assessed. Two methods were used to apply 40RNF to sugar beet seeds: incorporation into film-coats sprayed on to pre-pelleted seeds and incorporation into the pellet material prior to pelleting. Only 7.1% of applied 40RNF survived film-coating, but an initial concentration of 7 × 10⁸ ensured that 83.3% of a pre-determined target rate of 6 × 10⁷ |pellet was achieved. After 52 weeks of storage at 4°C,the numbers of 40RNF had declined by one to two orders of magnitude, with a decrease of approximately 50% in disease control. After 52 weeks at 18-20°C, 40RNF was below detectable limits (< 100|pellet), yet the biocontrol activity of the seed treatments was not reduced. The survival of 40RNF during incorporation into the pellet material was poor (< 0.2% of those applied, i.e. 5 × 10⁵ pellet). However, bacterial viability and biocontrol efficacy were maintained at 100% of the control value for 24 weeks when stored at 18-20°C. The results indicate that commercial seed treatments and the storage of pellets at ambient temperatures has potential for the introduction of bacterial BCAs into the spermosphere.     

Pelleting of broiler diets: An overview with emphasis on pellet quality and nutritional value

Pelleting is the most prevalent heat treatment in the production of poultry feed. The objective of pelleting is to agglomerate smaller feed particles into larger particles as pellets to enhance the economics of production by increasing the feed intake, and thus growth performance and feed efficiency. However, due to the heat, moisture and mechanical pressure applied during conditioning and pelleting, some chemical and physical alterations occur that may have beneficial or detrimental effects on feed components, gastrointestinal development and subsequent bird performance. Pelleting process has been shown to gelatinise starch, but only to a small extent, and thus may be of modest relevance in starch digestion. Pelleting process may also result in partial denaturation of proteins; a process which can potentially improve protein and to some extent starch digestibility due to inactivation of proteinaceous enzyme inhibitors. Cell wall breakage, as a result of the physical stress of pelleting, may also provide greater accessibility of nutrient contents, previously encapsulated within endosperm sub-aleurone, to digestive enzymes. In diets based on viscous cereals, nutrient availability may be negatively affected through increased digesta viscosity as a result of either an increase in soluble carbohydrate concentration or changes in the molecular weight of soluble fibres or both, due to pelleting. Pelleting process also remains a potentially aggressive process on the stability of exogenous feed enzymes and vitamins, a major concern of feed manufacturers. Particle size-reducing property of the pelleting process may result in a suboptimal gizzard development and thus reduced nutrient digestibility of diets for poultry. While physical pellet quality is a critical factor to optimise feed efficiency and growth response of broilers, the present review highlights that it is the balance between nutrient availability and physical quality of pellets which is critical in determining the actual performance of broilers. Under the conventional pelleting process, good pellet quality is usually obtained at the expense of nutritional quality. Research is warranted to identify and evaluate possible strategies to manufacture highly digestible high quality pellets. Such strategies will require novel approaches of improving feed hygiene which are not detrimental to feed nutrients.     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

Effects of grain type and conditioning temperature during pelleting on growth performance,ruminal fermentation,meat quality and blood metabolites of fattening lambs

Ruminants can tolerate moderate concentrations of dietary tannin, making it feasible to replace corn with sorghum in ruminant diets; however, conditioning temperature of pelleted total mixed ration (PTMR) greatly affects nutrient digestibility. The objective was to determine effects of grain type and conditioning temperature during pelleting on growth performance, ruminal fermentation, meat quality and blood metabolites of fattening lambs. This was a 2 × 3 factorial study, with corn and sorghum and three conditioning temperatures (65, 75 and 85 °C) in a randomized complete design, with 36 lambs (120 ± 10.2 d and 24.9 ± 3.3 kg) grouped by weight and randomly allocated. The resulting six PTMRs were referred to as 65-S, 75-S and 85-S for sorghum-based diets, and 65-C, 75-C and 85-C for corn-based diets, for low, medium and high pelleting temperatures, respectively. There was no grain type × conditioning temperature (Grain × Temp) interaction on growth performance and apparent nutrient digestibility. Furthermore, grain type did not affect DM intake (DMI), average daily gain (ADG) or feed conversion ratio (FCR) of fattening lambs. Pelleting at 75 °C improved ADG (P < 0.03) and FCR (P < 0.02) of fattening lambs compared to other temperatures. There was a Grain × Temp interaction (P < 0.01) on ruminal pH (lowest in lambs fed 75-S). There tended (P = 0.07) to be a Grain × Temp interaction for total volatile fatty acid (VFA), and there were Grain × Temp interactions for molar proportions of acetate (P < 0.04), butyrate (P < 0.03) and branch-chained VFA (P < 0.01). Lambs fed sorghum-based PTMR had greater molar proportion of propionate (P < 0.03) and lower acetate to propionate ratio (A:P, P < 0.04). Lambs fed sorghum-based PTMR had higher plasma concentrations of urea nitrogen (N) (P < 0.03), glucose (P < 0.01) and alkaline phosphatase (P < 0.05), whereas other blood metabolites were not affected by treatments. There were Grain × Temp (P < 0.03) interactions for color coordinates of longissimus and mid-gluteal muscle. Lambs fed sorghum-based PTMR had lower (P < 0.01) dressing percentage and meat quality than those fed corn-based PTMR. We concluded that sorghum can replace corn in lamb diets without compromising growth performance and feed efficiency; furthermore, feeding sorghum vs corn improved rumen fermentation, with reduced A:P ratio and enhanced N and glucose utilization. Finally, pelleting at 75 °C increased feeding value of either sorghum- or corn-based PTMR for fattening lambs.     

Afferent Modulation of Warmth Sensation and Heat Pain in the Human Hand

The hands of 14 normal humans were used to determine the somatotopic organization of the modulation of warmth sensation and heat pain by different forms of cutaneous stimuli. Test stimuli were 5-sec heat pulses ranging from 36° to 51°C, delivered to the fingerpads of digits 1, 2, 4, and 5 with a contact thermode. Conditioning stimuli (15 sec) bracketed the test stimuli and included vibration, noxious and innocuous heat, cold, and electrical pulses delivered to the fingerpads of digits that were adjacent or nonadjacent to the tested digits. Noxious (48° ± 1.3°C), but not innocuous (43°C), heat stimuli increased the perceived magnitude estimation of innocuous test stimuli (36–43°C) by 20–37% when delivered to adjacent, but not to nonadjacent, digits. No other conditioning stimuli had any effect on the intensity of warmth perception. In contrast, both noxious and innocuous heat or electrical conditioning reduced the magnitude estimation of noxious (50–5°C), but not innocuous, test pulses by 12–22% when delivered to adjacent digits. Conditioning of nonadjacent digits was significantly less effective. The analgesic effects of noxious and innocuous conditioning were approximately equal. Vibratory (120 Hz, 3.5 μm) and cold (15°C) conditioning stimuli were ineffective. The results are consistent with a dermatomal somatotopic organization of tactile and heat modulatory influences on warmth sensation and heat pain. The results further suggest that the neural mechanisms subserving warmth mediate a negative feedback influence on heat pain intensity.     

The role of energy in hyperthermia-induced mammalian cell inactivation: A study of the effects of glucose starvation and an uncoupler of oxidative phosphorylation

When cultured Chinese hamster cells were exposed to 43°C hyperthermia, effects due to glucose deprivation and to the presence of the uncoupler of oxidative phosphorylation, carbonylcyanide-3-chlorophenylhydrazone, during the 43°C treatment proved to be strongly accelerated compared to the effects at normal temperature (37°C). This strongly indicates that the availability of energy plays an important role in the response of these cells to hyperthermia. One of the reasons cells die after hyperthermia may be a lethal lack of energy. Cells heated before glucose deprivation were able to maintain viability for a longer period during deprivation than cells without the preheat treatment. As the cells might develop thermotolerance after the heat exposure, this suggests that cells in the thermotolerant state use energy in a more economical way.     

Effect of temperature on development and growth of the raptorial cladoceran Leptodora kindtii under laboratory conditions

1. Leptodora is a key species in many temperate freshwater systems, but so far its role in the food web could not be properly evaluated because detailed information about its secondary production was lacking. As we wanted to estimate the secondary production of Leptodora, we measured its development and growth rates in the laboratory. 2. Employing improved methods to estimate growth and instar durations, we cultured Leptodora kindtii in the laboratory at four constant temperatures (15, 17.5, 20 and 25 °C). Growth in length and development times of eggs and instar stages were assessed. 3. Growth rates at 15, 17.5 and 20 °C were similar, but at 25 °C growth was distinctly faster. At 17.5 °C we observed seven juvenile instar stages before the first adult instar stage was reached.     

Effect of abscisic acid on chilling injury of zucchini squash

The endogenous levels of abscisic acid (ABA) in zucchini squash were increased by temperature conditioning at 10°C for 2 days. This temperature conditioning treatment reduced the severity of chilling injury in the squash during subsequent storage at 2.5°C. The ABA levels remained higher in treated squash than in untreated samples throughout the storage. Direct treatments of squash with ABA at 0.5 and 1.0 mM before storage at 2.5°C increased ABA levels in the tissue and were also effective in reducing chilling injury.     

Evidence for the existence of cytoskeleton-bound polysomes in plants.

When conventional, high ionic strength buffers were used for the isolation of polysomes from pea plants, less than 20% were retained in the detergent-insoluble pellet. Reducing Tris, K+ and Mg++ to 10 mM increased retention to 70%, and when a new, microfilament-stabilizing buffer was used, retention increased to 80%. Conditions which favoured polysome pelleting at lower g forces permitted the retention of actin in the pellet. The data are consistent with the hypothesis that higher plants, like animals, contain cytoskeleton-(actin)-bound polysomes.     

Enhancing thermostability of <Emphasis Type="Italic">Escherichia coli</Emphasis> phytase AppA2 by error-prone PCR

Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60–80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6–7°C increases in melting temperatures (T _m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k _cat / K _m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.     

Comparative analysis of electricity cogeneration scenarios in sugarcane production by LCA

Purpose

The decentralization of the Brazilian electricity sector in association with the internal electricity supply crisis has encouraged companies in the sugarcane industry to produce electricity by burning sugarcane bagasse in cogeneration plants. This approach reduces the environmental impact of the sugarcane production and has opened up opportunities for distilleries and annex plants to increase their product portfolios. Potential scenarios for technically and environmentally improving the cogeneration performance were analyzed by using thermodynamic analysis and Life Cycle Assessment (LCA).

Methods

The method used in this study aimed to provide an understanding and a model of the electrical and thermal energy production and the environmental impacts of conventional vapor power systems which operate with a Rankine cycle that are commonly used by Brazilian distilleries. Vapor power system experts have suggested focusing on the following technical improvement areas: increasing the properties of the steam from 67 bar and 480 °C to 100 bar and 520 °C, regeneration, and reheating. Eight case scenarios were projected based on different combinations of these conditions. A functional unit of “To the delivery of 1.0 MWh of electricity to the power grid from a cogeneration system” was defined. The product system covers the environmental burdens of the industrial stage and the agricultural production of sugarcane.

Results and discussion

Technical evaluation indicated that the energy efficiency improves as the pressure at which the vapor leaves the boiler increases. Simultaneously, the net power exported to the grid increases and the makeup water consumption in the cooling tower and the makeup water supplied to the boiler reduce. From the LCA, it was noted that the improved energy performance of the system is accompanied by reduced environmental impacts for all evaluated categories. In addition, vapor production at 100 bar and 520 °C results in greater environmental gains, both in absolute and relative terms.

Conclusions

Reheating and regeneration concepts were found to be considerably effective in improving the energy and environmental performance of cogeneration systems by burning sugarcane bagasse. For the evaluated categories, the results indicate that the proposed modifications are favorable for increasing the efficiency of the thermodynamic cycle and for decreasing the environmental impacts of the product system.     

Cryopreservation of Plagiognathops microlepis sperm

Sperm was collected from cultured male fish and cryopreserved in 0.25 ml straws for the study of sperm cryopreservation. Different parameters were evaluated, including extender, dilution ratio, cryoprotectant type and concentration, equilibrium time, cooling height (in a two-step cooling protocol), and thawing temperature. The optimum result was obtained when the sperm was diluted at a 1:7 ratio in D-16 with 5% DMSO as a cryoprotectant, equilibrated for 20 min, held at 3 cm above liquid nitrogen for 10 min, and then stored in liquid nitrogen. After thawing in a water bath at 40 °C, the percentage of motile cells and fertilization rates of frozen-thawed sperm were 35.33 ± 2.52% and 39.00 ± 4.58%, respectively, while the corresponding rates for fresh sperm were 87.67 ± 3.06% and 88.67 ± 4.62%. We also used a programmed cooling protocol in which temperature was decreased from 4 °C to −80 °C by a rate of 30 °C/min, and then straws (0.25 ml) were placed above the surface of liquid nitrogen for 2 min before being stored in liquid nitrogen. This protocol provided a post-thaw activation rate of 36.67 ± 4.77%. Further parametric optimization is required to improve the quality of frozen-thawed sperm.     

A Rapid Method for Purification of d-Glucoside 3-Dehydrogenase

The stability of palm oil was tested by subjecting it to elevated temperatures for different durations of time, viz; at 80°C for 150 hr and 60°C for 400 hr.

The following results were obtained.

(1) The absorption spectrum resembled that of carotenoid and this changed progressively with a rise in peroxide and carbonyl values during the first 80 hr at 80°C.

(2) Peroxide values of Sabah palm oil were higher compared to Sumatra oil, there were marked increases in peroxide and carbonyl values of alkali refined oil as compared to crude oil. On the contrary, the residual color of crude Sumatra oil decreased considerably. Moreover, the steam emulsion number of alkali refined Sumatra oil was double the initial value after 400 hr.     

Prior temperature exposure affects subsequent chilling sensitivity

The chilling sensitivity of small discs or segments of tissue excised from chillingsensitive species was significantly altered by prior temperature exposure subsequent to holding the tissue at chilling temperatures as measured by a number of physiological processes sensitive to chilling. This temperature conditioning was reversible by an additional temperature exposure before chilling, and mature-green and red-ripe tomato tissue exhibit similar chilling sensitivities. Exposing pericarp discs excised from tomato fruit (Lycopersicon esculentum Mill. cv. Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h before chilling the discs at 2.5°C for 4 days significantly altered the rate of ion leakage from the discs, but had no effect on the rate of ion leakage before chilling and only a minimal effect on discs held at a non-chilling temperature of 12°C. Exposing chillingsensitive tissue to temperatures below that required to induce heat-shock proteins but above 20°C significantly increased chilling sensitivity as compared to tissue exposed to temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at 2.5°C were higher from fruit and vegetative tissue of chilling-sensitive species (Cucumis sativus L. cv. Poinsett 76, and Cucurbita pepo L. cv. Young Beauty) that were previously exposed for 6 h to 32°C than from similar tissue exposed to 12°C. Exposure to 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chilling tolerant species (Malus domestica Borkh. cv. Golden Delicious, Pyrus communis L. cv. Bartlett). Ethylene and CO₂ production were higher and lycopene synthesis was lower in chilled tomato pericarp discs that were previously exposed for 6 h to 32°C than the values from tissue exposed to 12°C for 6 h before chilling. Increased chilling sensitivity induced by a 6 h exposure to 32°C could be reversed by subsequent exposure to 12°C for 6 h.