结冷胶-黑米花青素-二氧化钛复合膜在蓝莓涂膜保鲜中的应用研究

以结冷胶、黑米花青素、二氧化钛为原料制备了可食性复合膜液,并利用复合膜液对蓝莓进行涂膜保鲜实验,研究新型复合膜对蓝莓的保鲜效果。首先,扫描电子显微镜发现结冷胶膜(GG)、结冷胶-黑米花青素复合膜(GG-BRA)和结冷胶-黑米花青素-二氧化钛复合膜(GG-BRA-TiO₂)的横截面无明显裂缝、无分层现象,傅里叶红外光谱进一步表明结冷胶可以通过非共价键与黑米花青素和二氧化钛进行相互作用。抑菌实验发现结冷胶-黑米花青素-二氧化钛复合膜(GG-BRA-TiO₂)具有良好的抑菌性,其中对金黄色葡萄球菌、大肠杆菌和面包酵母的抑菌圈分别为4.14、2.77和3.55mm。其次,利用复合膜液对蓝莓进行涂膜保鲜实验,结果表明GG-BRA和GG-BRA-TiO₂涂膜组能减缓蓝莓果实贮藏过程中的物理化学性质的变化速率,其中GG-BRA-TiO₂的延缓效果优于GG-BRA。经贮藏10 d,GG-BRA-TiO₂膜组的蓝莓失重率、硬度、花色苷、总酚、多酚氧化酶活力的下降分别较空白对照减少了1.56%、2...     

茶多酚-川陈皮素-羧甲基纤维素钠复合涂膜剂对刺嫩芽的保鲜效果

利用茶多酚、川陈皮素和羧甲基纤维素钠(Sodium carboxymethyl cellulose,CMC-Na)进行复合涂膜的制备,以刺嫩芽为保鲜对象,以感官评价、失重率、硬度、叶绿素、V_C含量、多酚氧化酶活性和丙二醛含量为指标,多方面评定复合涂膜对刺嫩芽的保鲜效果,并分析了可能的保鲜机理。结果表明:复合涂膜组对刺嫩芽的保鲜效果明显优于对照组,同时也明显优于茶多酚涂膜组和川陈皮素涂膜组。复合涂膜剂可有效地抑制刺嫩芽菜体褐变,维持多酚氧化酶和丙二醛处于较低水平,保持菜体组织形态,保证较高的感官品质,延缓其V_C含量和水分的损失。贮藏12 d时,与对照组相比,复合涂膜处理的刺嫩芽V_C含量提高1.4倍,失重率减少31%,综合各保鲜指标分析,复合涂膜可以延长刺嫩芽4~5 d的贮藏时间。     

可食性纳米TiO2-壳聚糖膜的制备及性能研究

以壳聚糖、苯甲酸钠、纳米二氧化钛为主要原料制备壳聚糖膜和可食性纳米TiO₂-壳聚糖复合膜，研究2种膜的性能及对鸡蛋保鲜效果的影响。结果表明：当苯甲酸钠浓度为0.4%、壳聚糖浓度为2.5%时，壳聚糖膜的性能最优，成膜效果最好；当纳米二氧化钛的添加量为2%时所制复合膜的性能最优。用制得的壳聚糖溶液和复合溶液对鸡蛋涂膜并进行理化性质检测，发现复合膜对鸡蛋有较好的保鲜效果。第14天时复合膜组的感官评价为浓蛋白较多，不散流，蛋黄呈扁球形，系带变细，鸡蛋气室高度为6.58 mm，失重率为7.23%，蛋黄指数为0.28，蛋清哈夫单位为72.4，蛋清pH值为8.63，鸡蛋总菌数为1.6×10⁴ cfu/mL。研究制备的复合膜对鸡蛋保鲜效果显著，为纳米二氧化钛在禽蛋保鲜领域的应用奠定了基础。     

壳聚糖/纳米TiO_2复合涂膜对芒果保鲜效果的影响

研究用壳聚糖单一涂膜及壳聚糖/纳米TiO_2复合涂膜对芒果保鲜效果的影响,并对不同纳米TiO_2添加量的复合膜进行微观形态观察。结果表明,当壳聚糖/纳米TiO_2复合膜中壳聚糖质量分数为1%,纳米TiO_2质量分数为0.03%时,涂膜处理可以保持芒果果实硬度、V_C及可滴定酸含量,延缓果胶的降解速度,保持细胞膜的功能活性。在贮藏第20 d时,芒果转黄指数为58.16%,失重率为6.72%,说明壳聚糖/纳米TiO_2复合涂膜对芒果在13℃贮藏条件下起到了较好的保鲜作用。     

次氯酸钠杀菌结合聚乙烯醇基纳米SiO_2复合材料涂膜对鸭蛋保鲜效果的影响

为探究次氯酸钠杀菌结合聚乙烯醇基纳米SiO_2复合材料涂膜对鸭蛋的保鲜效果,本实验分别采用次氯酸钠杀菌、聚乙烯醇基纳米SiO_2复合材料涂膜和次氯酸钠杀菌结合聚乙烯醇基纳米SiO_2复合材料涂膜处理新鲜鸭蛋。动态测定35 d贮藏过程中各组鸭蛋的失重率、菌落总数、哈夫单位和蛋黄指数的变化情况,比较贮藏后各组鸭蛋蛋壳色度和熟制鸭蛋质构变化。结果表明:与对照组相比,三个处理组鸭蛋的失重率和菌落总数均显著降低(p0.05),杀菌结合涂膜组的失重率和菌落总数最小,分别为2.79%±0.19%、(4.83±0.42)lg cfu/g;三个处理组鸭蛋的哈夫单位和蛋黄指数均显著增加(p0.05),杀菌结合涂膜组的哈夫单位(76.09±4.15)和蛋黄指数(0.40±0.02)最大,且与贮藏前的新鲜蛋无显著性差异(p0.05);杀菌结合涂膜处理对鸭蛋质构特性无显著影响(p0.05),但可使蛋壳亮度显著提高(p0.05)。由此可见,三种处理方式均能显著逆转鸭蛋品质的劣变,次氯酸钠杀菌结合聚乙烯醇基纳米SiO_2复合材料涂膜的保鲜效果最佳,显著优于单独的次氯酸钠杀菌和聚乙烯醇基纳米SiO_2复合材料涂膜(p0.05),该研究为杀菌结合复合材料涂膜在禽蛋保鲜上的应用提供参考。     

壳聚糖与纳米TiO_2对复合膜结构和性能的影响及复合膜的应用

以马铃薯淀粉为基材,壳聚糖和纳米TiO_2为增强相,共混制备马铃薯淀粉单膜(P)、马铃薯淀粉/壳聚糖复合膜(P+Ch)、马铃薯淀粉/纳米TiO_2复合膜(P+TiO_2)、马铃薯淀粉/壳聚糖/纳米TiO_2复合膜(P+Ch+TiO_2),对圣女果和青椒进行涂膜保鲜实验,通过测定贮藏期间圣女果和青椒的失重率、可溶性糖和可滴定酸含量的变化,评估复合涂膜对圣女果和青椒的保鲜效果,同时对四组复合膜的吸湿性、力学强度和保鲜效果进行比较。结果表明:壳聚糖、纳米TiO_2与马铃薯淀粉共混制膜具有良好的相容性; P+Ch+TiO_2的拉伸强度相比P、P+Ch、P+TiO_2分别提高了91.13%、77.78%、84.82%;吸湿性依次降低了60.91%、6.76%、49.45%。室温15℃存储30 d后,圣女果和青椒处理组的失重率均低于对照组,可溶性糖含量和可滴定酸含量高于对照组,处理组中P+Ch+TiO_2复合涂膜表现最佳保鲜效果。综上,壳聚糖和纳米TiO_2的添加改善了淀粉复合膜的性能,复合膜液对圣女果和青椒具有良好的保鲜效果。     

纳米TiO_2抗菌复合膜的制备及其在枇杷保鲜中的应用

为制备出综合性能较好的抗菌绿色包装材料用于枇杷保鲜。以明胶、羧甲基纤维素钠为成膜基材,添加具有抗菌活性的纳米TiO_2对复合膜进行改性处理,研究纳米TiO_2添加量对复合膜机械性能、阻隔性能和抑菌性能等方面的影响。通过膜的性能测试,确定最佳的纳米TiO_2添加量并将其应用于枇杷保鲜。研究表明:纳米TiO_2添加量为2%时,复合膜的机械性能、成膜特性及抗菌活性相对最好。该处理组枇杷的呼吸强度、失重率,木质化含量显著低于对照组(P0.05)。冷藏至35 d时,处理组枇杷的菌落总数、腐烂率及MAD含量显著低于对照组(P0.05)。该研究可为纳米TiO_2抗菌复合膜的制备及枇杷保鲜提供新参考。     

N、S改性TiO2及其与淀粉复合膜的抑菌性能及保鲜效果

本文采用脲、硫脲分别对TiO₂进行N、S掺杂改性,通过X-射线衍射和傅里叶红外光谱对改性后复合材料的晶形结构和化学键进行表征分析。研究了N、S改性TiO₂复合材料及其淀粉复合膜在可见光下的抑菌性能;通过测定贮藏期内圣女果腐烂率、失重率及营养物质含量的变化评价复合淀粉膜的保鲜效果。结果表明:N、S改性后的TiO₂复合材料对大肠杆菌的抑菌率分别达到89%和95%,相较于TiO₂分别提高了24%和30%。改性后的TiO₂复合材料能均匀分散在玉米淀粉膜中。将复合膜用于圣女果保鲜,与无膜组相比,经N-TiO₂淀粉复合膜处理的圣女果贮藏8 d后其腐烂率和失重率分别降低了3.33%和10.66%,V_C含量、总酸含量和总糖含量分别是无膜组的1.08、1.06、1.03倍;经S-TiO₂淀粉复合膜处理的圣女果在贮藏8 d后,其腐烂率和失重率分别降低了6.25%和10.90%,V_C含量、总酸含量和总糖含量分别是无膜组的1.11、1.12、1.08倍。综合分析,N-TiO₂、S-TiO₂淀粉复合膜均可有效抑制圣女果贮藏期间的腐烂率、失重率及营养物质的散失,维持其采后新鲜度,延长保鲜期。     

壳聚糖/CMC复合涂膜处理对柑橘果实采后品质的影响

流延法制备壳聚糖单层膜及羧甲基纤维素钠/壳聚糖复合膜,并测定膜机械性能、微观结构和水蒸气透过性等对其进行表征;以春香和柠檬两个柑橘品种为试材,研究壳聚糖单层涂膜及羧甲基纤维素钠/壳聚糖自组装复合涂膜对两种柑橘果实（春香,柠檬）采后货架期品质的影响。结果发现：复合膜的水蒸气透过性显著（p<0.05）高于单层膜,为壳聚糖单层膜的1.74倍;且维持良好的力学性能及微观结构。应用于柑橘采后保鲜,发现涂膜可提高柑橘果亮度,抑制抗坏血酸氧化,减少硬度降低。涂膜处理可减少柑橘果实表面气孔数量,降低贮藏期间柑橘果实失重率,贮藏20 d处理组失重率均在7.5%以下;且涂膜延缓了柑橘可滴定酸含量的下降。与单层涂膜相比,羧甲基纤维素钠/壳聚糖自组装复合涂膜可显著（p<0.05）提高春香、柠檬硬度及抗坏血酸含量。经20 d贮藏,复合涂膜组的春香及柠檬硬度分别是对照组的1.15倍、1.23倍。复合膜证实了静电吸附自组装形成的多糖基膜具有维持柑橘采后品质的潜力,为开展不同柑橘品种生物涂膜保鲜研究提供参考。     

壳聚糖及其与纳米SiO_x复合涂膜对莲藕贮藏品质的影响

配制了壳聚糖、壳聚糖/纳米SiOx和壳聚糖/纳米SiOx/氯化亚锡3种涂膜液,对莲藕进行了涂膜,以不涂膜莲藕为对照,置于0～10℃贮藏,通过测定失重率、呼吸强度、L值、PPO活性、可溶性糖质量分数及硬度,研究了壳聚糖及其复合膜对莲藕贮藏保鲜过程中品质的影响.结果表明:壳聚糖/纳米SiOx组效果最好,有效地抑制了莲藕采后呼吸、失重、糖物质消耗、PPO活性升高、颜色变暗及硬度下降,而壳聚糖/纳米SiOx膜中添加氯化亚锡并不能对莲藕保鲜效果起到更好的作用.     

纳米二氧化钛涂覆聚对苯撑苯并二噁唑纤维的制备及其抗紫外光照射性能

针对聚对苯撑苯并二噁唑(PBO)纤维经紫外光照射后强力下降的问题,首先采用氧等离子体对PBO纤维表面进行改性,提高其界面性能;然后在改性PBO纤维表面涂覆纳米TiO₂及有机硅整理剂制备TiO₂/PBO复合纤维;最后对复合纤维的结构和性能进行表征与分析。结果表明:当氧等离子体处理功率为200 W、处理时间为200 s时,PBO纤维表面有凹痕,纤维拉伸强力保持率大于90%,摩擦因数增大16%,接触角减小为52.7°,说明PBO纤维的表面润湿性能增大;当纳米TiO₂与硅烷偶联剂质量比为1∶1时,TiO₂/PBO复合纤维表面有沉积凸起的纳米TiO₂颗粒;用紫外光照射200 h后,TiO₂/PBO复合纤维的断裂强力下降率比PBO原纤减少30%,说明纳米TiO₂涂覆后的PBO纤维抗紫外光照射性能提高。     

海藻酸钠/纳米TiO2复合涂膜对采后番木瓜果实软化及细胞壁降解影响

为探究海藻酸钠/纳米TiO₂(sodium alginate/nano-TiO₂,SA/TiO₂)复合涂膜处理抑制采后番木瓜果实软化的作用机理,以“日升10号”番木瓜为试材,采用SA/TiO₂复合涂膜处理,研究采后番木瓜果实硬度、细胞壁组分含量和细胞壁水解酶活性的变化规律。结果表明,SA/TiO₂复合涂膜处理能有效地抑制采后番木瓜果实中多聚半乳糖醛酸酶(polygalacturonase,PG)、果胶甲酯酶(pectin methylesterase,PME)、纤维素酶(cellulase,Cx)和β-半乳糖苷酶(β-D-galaetosidase,β-Gal)活性,减缓原果胶和纤维素的水解,减少可溶性果胶的增加,从而保持较完整的细胞壁结构,维持果实硬度。因此认为,SA/TiO₂复合涂膜处理可延缓采后番木瓜果实的软化进程,提高果实耐贮性。     

Efficacy of cellulose-based coating on enhancing the shelf life of fresh eggs

Methylcellulose (2.00% w/v) and hydroxypropyl methylcellulose (1.00% w/v) -based coating was formulated to study the effects of polyethylene glycol-400 (PEG-400) and a stearic and palmitic acid blend (SPB) on water vapour permeability (WVP) and tensile properties. The efficacy of cellulose-based coating on fresh egg quality during 28-day storage at ambient temperature was investigated in terms of weight loss, pH, and albumen quality. The selected formulation obtained by a response surface analysis was 1.00% w/v PEG-400 and 4.00% w/v SPB. An edible cellulose-based solution was prepared for eggshell coating. A batch of fresh, grade AA 1-day eggs was coated with cellulose-based coating solution. The other batch consisted of uncoated eggs, which served as the control. Lower weight loss (4.28%) was observed in the cellulose-based coated eggs, compared to 8.83% for the uncoated eggs. The pH in albumen of coated and uncoated eggs increased from 8.71 and 8.72 to 9.44 and 9.76, respectively, after 4 weeks of storage. For albumen quality, Haugh units indicated that after 7 days, cellulose-based coated eggs changed from grade AA to grade A, and remained in grade A throughout the storage period; whereas after 5 days, uncoated eggs started to change from grade AA to grade A, and continually degraded to grade B during the 4 weeks of storage. This study highlights the promising use of cellulose-based coating to enhance the shelf life of fresh eggs.     

Edible Film Coating to Minimize Eggshell Breakage and Reduce Post-Wash Bacterial Contamination Measured by Dye Penetration in Eggs

ABSTRACT: The mechanical and bacterial barrier properties of washed (tap water, sodium carbonate, Na₂CO₃, sodium hypochlorite, NaOCl) noncoated eggs and eggs coated with soy protein isolate (SPI), whey protein isolate (WPI), carboxylmethyl cellulose (CMC), or wheat gluten (WG) were evaluated. All coated eggshells showed greater puncture strength than those of noncoated eggs. The film-coated eggs reduced post-wash bacterial penetration as measured by dye penetration method. WPI coating completely inhibited blue dye penetration on average. These results suggest that WPI coatings can be used to reduce breakage of eggshell and egg microbial contamination.     

纳米SiO2改性聚偏二氯乙烯涂膜对咸鸭蛋品质的影响

以共混法制备纳米SiO2改性聚偏二氯乙烯（polyvinylidene chloride,PVDC）基复合涂膜材料并研究其对咸鸭蛋品质变化的影响。结果表明,与未涂膜组相比,纳米SiO2/TiO2交联改性聚乙烯醇（polyvinyl alcohol,PVA）基膜材料、纳米蒙脱土（montmorillonite,MMT）改性PVA基膜材料、PVDC膜材料以及纳米SiO2改性PVDC基膜材料均能显著减少咸鸭蛋在贮藏过程中的不良品质变化（质量损失率增加、挥发性盐基氮（total volatilebase nitrogen,TVB-N）含量升高、菌落总数增加）（P＜0.05）。SiO2改性PVDC膜材料涂膜处理对咸鸭蛋质量损失率、菌落总数、TVB-N和游离脂肪酸的累积以及蛋青蛋黄中pH值变化的抑制程度显著高于其他涂膜处理组（P＜0.05）。另外,SiO2改性PVDC膜材料能显著抑制咸鸭蛋中菌落总数的增长（P＜0.05）,并维持产品的感官品质（P＜0.05）。在28 ℃、相对湿度70%条件下,与未涂膜组相比较,SiO2改性PVDC膜材料能够延长咸鸭蛋的贮藏保质期70 d（P＜0.05）,该研究结果为咸鸭蛋的保鲜运输及贮藏提供了理论依据。     

Effect of bilayer coating composed of polyvinyl alcohol,chitosan, and sodium alginate on salted duck eggs

The composite bilayer film based on polyvinyl alcohol, sodium alginate, and chitosan were developed in this study, and the effects of the related coating on internal-quality changes of salted duck eggs (SDEs) during storage were determined. The results showed that the overall properties of the bilayer film were better than those of monolayer films, including storage stability. Coatings could significantly (p < 0.05) reduce quality deterioration of SDEs such as water loss, oil exudation, and gritty. The bilayer coating was more effective in inhibiting the increase of egg yolk pH and total bacterial counts than monolayer coatings. After storage for 60 days, water content of bilayer coated SEDs was 19.7% higher than that of the control SDEs, oil exudation was 38% higher, and gritty texture was 27.9% higher. Moreover, bilayer coating could prolong the shelf life of SDEs to more than 60 days at 25°C and 50% relative humidity according to the results of total bacterial count experiment, while the shelf life of control SDEs was less than 45 days. This study indicated the potential application of bilayer composite coating for preservation of SDEs.     

Oil Coating Affects Internal Quality and Sensory Acceptance of Selected Attributes of Raw Eggs during Storage

Four (coconut, palm, rice bran, and soybean) edible oils and glycerol were applied on eggshell. All noncoated and coated eggs were stored for 5 wk at 25 ± 2 °C and drawn weekly for quality evaluation. All oil coatings were more effective in preserving internal quality of eggs than was glycerol coating. As storage time increased, the preservative effects of edible oil coating on weight loss, and albumen and yolk quality were significantly noticed. Oil‐coated eggs had significantly lower weight loss (<0.43%) than did noncoated (3.87%) and glycerol‐coated (3.73%) eggs after 5 wk of storage. Based on the Haugh unit, oil‐coated eggs maintained AA grade up to 3 wk. After 5 wk of storage, noncoated, glycerol‐coated, and oil‐coated eggs changed from AA grade to below B, below B and A grade, respectively. The albumen pH of noncoated and glycerol‐coated eggs considerably increased from 8.23 to 9.51 and 9.42, respectively, while those of oil‐coated eggs either maintained or slightly increased to 8.32. The albumen viscosity of all eggs decreased with increased storage time. Consumers (N = 120) could differentiate surface glossiness of oil‐coated eggs from uncoated eggs (R‐index of 81.42% to 86.99%). All oil‐coated eggs were acceptable for surface glossiness (liking scores of 6.22 to 6.77) and surface odor (liking scores of 6.20 to 6.55) with overall liking scores of 6.34 to 7.03. Overall, this study demonstrated that edible oil (coconut, palm, rice bran, and soybean) coating could preserve internal quality of eggs (maintaining grade A) at least 4 wk longer than noncoated eggs. Practical Application: Freshness is a major contribution to the egg quality. The internal quality of eggs begins to deteriorate after they have been laid due to loss of moisture and carbon dioxide via the eggshell pores. Refrigeration is very effective in preserving egg quality. Surface coating is an alternative method to preserve egg quality, although it is much less effective than refrigeration. This study demonstrated that coconut, rice bran, soybean, and palm oils, which are abundant and commonly consumed in many parts of the world, could preserve the internal quality and reduce weight loss of oil‐coated eggs during room temperature storage.     

海藻酸钠和羧甲基纤维素钠涂膜改善鲜切豇豆贮藏特性的比较分析

为了提高鲜切豇豆的品质,本文研究了海藻酸钠(0.6%,m/m)和羧甲基纤维素钠(0.8%,m/m)两种涂膜剂对其贮藏期间的呼吸强度、维生素C、叶绿素等品质指标变化规律的影响。研究发现,与对照相比,在贮藏期间,海藻酸钠和羧甲基纤维素钠涂膜均有效降低鲜切豇豆的呼吸强度,维持较高的可溶性固形物含量,减少鲜切豇豆维生素C和叶绿素的损失,保持较高的过氧化物酶活性,减少丙二醛的积累。贮藏至12 d,海藻酸钠涂膜的鲜切豇豆呼吸强度为237.67 mg/(kg·h),处于较低水平;其维生素C、叶绿素及可溶性固形物含量分别比羧甲基纤维素钠涂膜的高13.06 mg/100 g、0.62 mg/g、0.49%,但丙二醛含量低11.36%。研究结果表明,海藻酸钠涂膜更有利于保持鲜切豇豆的品质及延长贮藏期,其用于豇豆的保鲜贮藏具有潜在的应用前景。     

Edible coatings for enhancing microbial safety and extending shelf life of hard-boiled eggs

ABSTRACT:  Hard-boiled eggs were coated with chitosan–lysozyme (CL), whey protein isolate (WPI), or Bake sheen (BS), inoculated with Listeria monocytogenes or Salmonella enterica Ser. Enteritidis at 10⁴ CFU/g, and stored for 4 wk at 10 °C. Microbial populations were enumerated weekly. Two nonchallenge studies were also conducted with hard-boiled eggs: coated shell-on eggs packaged in plastic containers or peeled eggs coated and vacuum-packaged. Total plate counts (TPC), coliforms, yeasts, molds, weight loss, pH, and color of eggs were determined during the 10-wk storage at 10 °C. Uncoated eggs served as controls. All the coatings were not effective in inhibiting the growth of L. monocytogenes, but CL coating controlled the growth of S. Enteritidis. At the end of 4-wk storage, the numbers of S. enteritidis on CL-coated eggs were about 4-log₁₀ CFU/g less than that of the controls. Coatings reduced ( P < 0.05) the populations of coliforms and TPC, and completely inhibited mold growth during the 10-wk storage. Coatings also reduced ( P < 0.05) the weight loss of eggs, 4.1% to 4.8% on coated eggs compared with 7.5% in uncoated ones at the end of 10-wk storage. The pH of CL-coated eggs remained stable throughout the storage period, while the control eggs increased from 7.6 to 8.6. Color changes in CL- and WPI-coated eggshells were less ( P < 0.05) than those of BS-coated and the control. The CL coating effectively suppressed the numbers of TPC, coliforms, yeasts, and molds on peeled eggs during the 6-wk storage ( P < 0.05). The results suggest that CL coating on hard-boiled eggs can control the growth of S. Enteritidis and reduce undesirable changes in the interior quality of eggs.     

Separation of Egg White Lysozyme by Anionic Polysaccharides

Anionic polysaccharides (Na-alginate, Na-carboxyl-methyl-cellulose, high methoxy pectin and κ-carrageenan) were evaluated for their suitability towards simple and more efficient separation of lysozyme from salted or fresh duck eggs and hen egg whites for use in foods. Only κ-carrageenan interacted and formed precipitates with lysozyme. Recovery of lysozyme from fivefold diluted salted duck egg whites was 60–65% and 78–81% from fresh duck and hen egg whites using 0.7%κ-carrageenan. The recovered lysozyme from the salted duck egg whites was stable during storage at 4°C for 35 days.