南极磷虾（Euphausia superba）蛋白质深加工新技术研究
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摘要
南极磷虾资源量巨大,含有的蛋白质丰富质优,但是,其氟含量异常高,且含有功能强大的内源酶。因此,针对南极磷虾的这些特点,对其蛋白质进行深加工研究,从而开发安全高值的产品具有重要的意义。本文研究了南极磷虾蛋白质深加工的两套新技术：一套是基于低氟南极磷虾蛋白质基料制备的综合利用技术,包括低氟南极磷虾蛋白质基料的研究,利用低氟南极磷虾蛋白质基料制备蛋白粉的研究,以及低氟南极磷虾蛋白质基料制备过程中产生的废液副产物利用的研究；另一套是基于利用南极磷虾二段自溶制备高氨基酸态氮自溶液的技术,包括南极磷虾二段自溶技术的研究,及其自溶液的氟脱除和风味成分的研究。本文的主要研究内容和结论如下。
1.系统研究了碱溶酸沉法提取南极磷虾蛋白质的工艺,在此基础上研究了酸沉蛋白质的氟脱除技术,制备了低氟南极磷虾蛋白质基料,并对其进行了分析评价。结果表明,磷虾蛋白质溶解的适宜条件为pH11.5和4℃；适宜的蛋白质溶解方法是二次碱溶：第一次碱溶和第二次碱溶的加水比分别为6(水/磷虾,mL/g)和3(水/沉淀物,mL/g),时间均为30min,此方法下蛋白质溶出率为97.23%；最适的酸沉pH值是4.6；酸沉过程中,合理应用谷氨酰胺转氨酶能够使蛋白质得率提高5%；酸沉蛋白质经过一种氟脱除技术的处理,能够制备氟含量低于2mg/kg的低氟南极磷虾蛋白质基料,其蛋白质得率是52.68%；低氟南极磷虾蛋白质基料含有33.01%(以干基计)的总脂和66.96%(以干基计)的粗蛋白,其脂肪酸富含EPA (13.65%)、DHA (7.17%)和MUFAs (16:18.70%,18:120.61%),其蛋白质含有WHO/FAO/UNU要求的全部必需氨基酸,蛋白质中的每一项必需氨基酸含量均高于WHO/FAO/UNU对成人(婴儿)的要求。
2.对利用低氟南极磷虾蛋白质基料制备蛋白粉进行了研究。结果表明,喷雾干燥制备蛋白粉时,部分前处理的适宜条件是控制进料干物质含量≥7.89%,调节物料至pH7.2,40Mp高压均质,进料液温度为60℃；制备的蛋白粉水分含量<5%,其总脂含量高、磷脂含量高和表面游离脂肪含量高；喷雾干燥过程中,较低的进料干物质含量导致液滴固化后颗粒表层蛋白质的含量较大,蛋白粉的变性程度增加,而进料干物质含量的增加导致液滴固化后脂质更易滞留在颗粒表面,蛋白质更倾向于被包埋在颗粒内部；制备的低氟南极磷虾蛋白粉在空气下容易氧化,充氮包装能够良好地抑制其氧化；充氮包装下,添加抗氧化剂的低氟南极磷虾蛋白粉的抗氧化性显著地(P<0.05)提高,其中,一种复合抗氧化剂(L一抗坏血酸棕榈酸酯+dl-α-醋酸生育酚,1+1)的效果最优。
3.低氟南极磷虾蛋白质基料制备过程中产生了两股废液副产物：酸沉废液和脱氟废液。本文研究了废液副产物的利用,即研究向废液副产物中添加南极磷虾,利用二段自溶技术制备高氨基酸态氮南极磷虾自溶液的效果。结果表明,酸沉废液和脱氟废液的含氮成分主要是小分子肽或氨基酸；在利用酸沉废液时,通过二段自溶技术制备的自溶液的氨基酸态氮达到1.070g/100mL,总氮达到1.97g/100mL,铵盐占氨基酸态氮的比例为21.18%,氟含量为18.85μg/mL,蛋白质利用率达到79.48%；在利用酸沉废液和脱氟废液的混合物时,通过二段自溶技术制备的自溶液的氨基酸态氮达到1.026g/100mL,总氮达到1.94g/100mL,铵盐占氨基酸态氮的比例为19.71%,氟含量为18.79μg/mL,蛋白质利用率达到77.32%。
4.对南极磷虾二段自溶技术进行了研究,即研究向南极磷虾中添加水,利用二段自溶技术制备高氨基酸态氮南极磷虾自溶液的效果。此外,研究了南极磷虾一段自溶的部分影响因素。
南极磷虾一段自溶影响因素的研究结果表明,合适浓度的Na+、K+、Ca2+、 Mg2+溶液对磷虾自溶均有显著的(P<0.05)促进作用,而Zn2+溶液对磷虾自溶的促进作用不显著(P>0.05),较高浓度的Zn2+溶液对磷虾自溶还具有显著的(P<0.05)抑制作用；适度的紫外线辐照预处理可使磷虾一段自溶液的氨基酸态氮含量达到0.739g/100mL。
南极磷虾二段自溶技术的研究结果表明,南极磷虾二段自溶时,第二段自溶的最优条件是：液料比2.23,初始pH7.24,温度40.9℃,时间12h。最优的二段自溶条件下制备的二段自溶液含有氨基酸态氮0.951g/100mL、总氮1.92g/100mL,这两者均高于国家标准《酿造酱油》GB18186-2000对低盐固态发酵酱油特级品的相应指标值要求；二段自溶液含有的铵盐占氨基酸态氮的比例符合国家标准《酿造酱油》(征求意见稿)GB18186的新规定,含有的氟为18.60μg/mL；二段自溶液的蛋白质利用率达到75.33%。南极磷虾二段自溶技术具有蛋白质利用率高、工序简单、设备投入小、生产周期短、自溶液的氨基酸态氮含量高的优点；制备的二段自溶液含有WHO/FAO/UNU要求的全部必需氨基酸,其中Trp、Thr、His和Phe+Tyr是限制性氨基酸。二段自溶技术大幅度提高了自溶液的鲜味氨基酸含量,二段自溶液的总鲜味氨基酸达到46.06mg/mL,比一段自溶液的总鲜味氨基酸提高了23.71mg/mL,提高率达到106.09%。
5.对南极磷虾二段自溶液的氟脱除进行了研究。结果表明,采用钙盐沉淀法对南极磷虾自溶液脱氟时,在氯化钙、乳酸钙和醋酸钙中,以乳酸钙的氟脱除效果最优；乳酸钙作用下的初步脱氟液含有氟8.19g/100mL,氨基酸态氮0.854g/100mL,总氮1.72g/100mL,其总氮损失率是9.54%；在应用乳酸钙脱氟的基础上,一种生物脱氟剂能够将南极磷虾自溶液的氟含量降低至1.53mg/L,从而解决了南极磷虾自溶液中氟的安全性问题；该生物脱氟剂作用下的深度脱氟液含有氟1.53g/100mL,氨基酸态氮0.783g/100mL,总氮1.67g/100mL,其总氮损失率是14.06%。
对南极磷虾二段自溶液的风味成分进行了研究。结果表明,2-甲基丁醛、3-甲基丁醛、3-甲基-1-丁醇、乙醇是南极磷虾自溶液的主体风味成分,其相对丰度分别为24.56%、17.79%、17.70%和5.14%；南极磷虾自溶液的总挥发性风味化合物中,2-甲基丁醛、3-甲基丁醛、3-甲基-1-丁醇这三种食品香料的总相对丰度高达60.05%。
The biomass of Antarctic krill is huge, and Antarctic krill are rich in proteins with high quality. But, the fluoride content of Antarctic krill is extremely high and Antarctic krill contains very strong proteolytic enzymes. Thus, based on these characters of Antarctic krill, the investigation of deep processing of Antarctic krill proteins is important to develop safty krill commodities wih high added value. Two sets of new technologies for deep processing of Antarctic krill proteins were investigated in this thesis. One was based on the preparation of proteins with low fluoride level from Antarctic krill, including the preparation of proteins with low fluoride level from Antarctic krill, the preparation of protein powders by using the proteins with low fluoride level, and the utilization of the byproducts of waste liquor produced during the preparation of proteins with low fluoride leve. Another was the preparation of hydrolysates with high amino acid nitrogen level by using the technology of two-stage krill autolysis, including the investigation of the technology of two-stage krill autolysis, and the investigation of the fluoride removal and volatile flavor compounds of hydrolysates obtained by two-stage krill autolysis. The main investigations and their results were as follows.
1. The alkali solubilization and acid precipitation method for recovering proteins from Antarctic krill was systematacially investigated. Based on the investigation of the method, the fluoride removal technology of the acid-precipitated proteins obtained by the method was investigated, and the proteins with low fluoride level were prepared. Besides, the composition of the proteins with low fluoride level was analyzed. The optimal conditions for protein solubilization were determined to be pH11.5and4℃. The optimum method of protein solubilization was the proteins were solubilized two times; a water/krill ratio (mL/g) of6and a time of30min were used for the first step, whereas the second used a water/krill residue ratio (mL/g) of3and a time of30min. The dissolved protein rate by the optimum method of protein solubilization was97.23%. The optimum pH for protein precipitation was4.6. The protein recovery yield was increased by5%when transglutaminase was correctly applied during the acid-induced protein precipitation. The proteins with low fluoride level were finally obtained through an effective fluoride removal technology of the acid-precipitated proteins. The fluoride content of the proteins with low fluoride level was lower than2mg/kg. The protein yield of the proteins with low fluoride level was52.68%. The proteins with low fluoride level were composed of66.96%of crude proteins (dry weight) and33.01%of total lipids (dry weight). The fatty acids of the proteins with low fluoride level were rich in EPA(13.65%)、DHA(7.17%)和MUFAs (16:18.70%,18:120.61%). The proteins with low fluoride level contained all essential amino acids in sufficient amounts to meet WHO/FAO/UNU requirements for adults and infants.
2. The preparation of protein powders by using the proteins with low fluoride level was investigated. The optimal conditions of some pretreatments of spray drying were determined as follows:the dry matter content of feeds (≥7.89%), the adjusted pH of materials (7.2), the high pressure of homogenization (40Mp), the temperature of feeds (60℃). The moisture content of the prepared protein powders was lower than5%, and the protein powders contained high levels of total lipids, total phospholipids and surface free-fat. During the spray drying process, a lower dry matter content of feeds resulted in a higher protein content on the particle surface after the liquid drops were solidified, which made the denaturation degree of protein powders increase, whereas an increased dry matter content of feeds led to more lipids existing on the particle surface and more proteins enclosed in the particle interior after the liquid drops were solidified. The prepared protein powders with low fluoride level exhibited a bad oxidation resistance when they were exposed to air, and their easy oxidation properties were well inhibited by nitrogen-filled packages. Under the condition of nitrogen-filled packages, the antioxidant properties of the protein powders with antioxidant addition were significantly (P<0.05) improved, and a combined antioxidant (L-ascorbyl palmitate+DL-a-tocopheryl acetate,1+1) exhibited the best antioxidant performance.
3. The preparation of the proteins with low fluoride level resulted in two waste liquor byproducts, of which one was produced during the acid-induced protein precipitation, and another was produced during the fluoride removal of the acid-precipitated proteins. The utilization of the byproducts was investigated through the addition of Antarctic krill to the byproducts, followed by the preparation of hydrolysates with high amino acid nitrogen level using the technology of two-stage krill autolysis in this thesis. The main nitrogen-containing ingredients of waste liquor byproducts were small molecules of peptides or amino acids. Through the utilization of waste liquor produced during the acid-induced protein precipitation, the hydrolysates obtained by two-stage krill autolysis contained1.070g/100mL of amino acid nitrogen,1.97g/100mL of total nitrogen,18.85μg/mL of fluoride, meanwhile the ammonium salt to amino acid nitrogen rate and protein yield of the hydrolysates were21.18%and79.48%, respectively. Through the utilization of the mixture of waste liquor produced during the acid-induced protein precipitation and the fluoride removal of the acid-precipitated proteins, the hydrolysates obtained by two-stage krill autolysis contained1.026g/100mL of amino acid nitrogen,1.94g/100mL of total nitrogen,18.79μg/mL of fluoride, meanwhile the ammonium salt to amino acid nitrogen rate and protein yield of the hydrolysates were19.71%and77.32%, respectively.
4. The technology of two-stage krill autolysis was investigated. During the investigation, distilled water was added to Antarctic krill, and the preparation of hydrolysates with high amino acid nitrogen level using the technology was investigated. Furthermore, the partial influence factors of one-stage krill autolysis were investigated.
The investigation of the influence factors of one-stage krill autolysis indicated Na+, K+, Ca2+and Mg2+solutions at appropriate concentrations showed significantly (P<0.05) promoting effects on krill autolysis, respectively, and the promoting effect of Zn2+solution on krill autolysis was insignificant (P>0.05). Furthermore, the Zn2+solution at high concentration exhibited significantly (P<0.05) inhibitory effects on krill autolysis. The amino acid nitrogen content of hydrolysates obtained by one-stage krill autolysis was0.739g/100mL through the pretreatment of appropriate ultraviolet radiation.
The technology of two-stage krill autolysis was investigated. The optimal conditions of the second-stage krill autolysis during two-stage krill autolysis were determined to be a liquor to material rate of2.23(g/g), an initial pH of7.24, a temperature of40.9℃, a time of12h. Under these optimal conditions, the hydrolysates obtained by two-stage krill autolysis contained1.026g/100mL of amino acid nitrogen and1.92g/100mL of total nitrogen, and each of them was in sufficient amount to meet Chinese National Standard GB18186-2000requirements for the special grade soy sauce of low salt-solid fermentation. The ammonium salt to amino acid nitrogen rate of the hydrolysates met the new requirement of Chinese National Standard GB18186(Draft for Comment) for fermented soy sauce, and the fluoride content and protein yield of the hydrolysates were18.60μg/mL and75.33%, respectively. The technology of two-stage krill autolysis had the advantages of high protein yield, simple treatment processes, low equipment investment, short processing time, and prepared hydrolysates with high amino acid nitrogen level. The prepared hydrolysates contained all essential amino acids required by WHO/FAO/UNU, of which Trp, Thr, His and Phe+Tyr were the limiting amino acids. The technology of two-stage krill autolysis could greatly improve the total flavour amino acid content of the hydrolysates, which was46.06mg/mL. The total flavour amino acid content of hydrolysates obtained by two-stage krill autolysis increased by23.71mg/mL compared to that of hydrolysates obtained by one-stage krill autolysis, and the increasing rate was106.09%.
5. The fluoride removal of hydrolysates obtained by two-stage krill autolysis was investigated. Compared to calcium chloride and calcium acetate, calcium lactate exhibited the best fluoride removal efficiency when the calcium precipitation method was employed to remove fluoride from the hydrolysates. The solution obtained by the preliminary fluoride removal of the hydrolysates through the application of calcium lactate contained8.19μg/mL of fluoride,0.854g/100mL of amino acid nitrogen,1.72 g/100mL of total nitrogen, and the loss rate of total nitrogen was9.54%. Baed on the preliminary fluoride removal of the hydrolysates through the application of calcium lactate, a biological defluorination agent could efficiently remove the residuaful fluoride from the hydrolysates and make the fluoride content of the hydrolysates decrease to1.53μg/100mL. Therefor, the safty problem of fluoride in the hydrolysates was successfully resolved. The solution obtained by the deep fluoride removal of the hydrolysates through the application of the biological defluorination agent contained1.53μg/mL of fluoride,0.783g/100mL of amino acid nitrogen,1.67g/100mL of total nitrogen, and the loss rate of total nitrogen was14.06%.
The volatile flavor compounds of hydrolysates obtained by two-stage krill autolysis were investigated. The main volatile compounds of the hydrolysates were identified as2-methyl-Butanal,3-methyl-Butanal,3-methyl-1-Butanol, Ethanol, and each of their relative abundance in the identified volatiles was24.56%,17.79%,17.70%and5.14%, respectively. The three compounds of2-methyl-Butanal,3-methyl-Butanal and3-methyl-1-Butanol are permited to use as food flavors, and their total relative abundance was60.05%.
1.系统研究了碱溶酸沉法提取南极磷虾蛋白质的工艺,在此基础上研究了酸沉蛋白质的氟脱除技术,制备了低氟南极磷虾蛋白质基料,并对其进行了分析评价。结果表明,磷虾蛋白质溶解的适宜条件为pH11.5和4℃；适宜的蛋白质溶解方法是二次碱溶：第一次碱溶和第二次碱溶的加水比分别为6(水/磷虾,mL/g)和3(水/沉淀物,mL/g),时间均为30min,此方法下蛋白质溶出率为97.23%；最适的酸沉pH值是4.6；酸沉过程中,合理应用谷氨酰胺转氨酶能够使蛋白质得率提高5%；酸沉蛋白质经过一种氟脱除技术的处理,能够制备氟含量低于2mg/kg的低氟南极磷虾蛋白质基料,其蛋白质得率是52.68%；低氟南极磷虾蛋白质基料含有33.01%(以干基计)的总脂和66.96%(以干基计)的粗蛋白,其脂肪酸富含EPA (13.65%)、DHA (7.17%)和MUFAs (16:18.70%,18:120.61%),其蛋白质含有WHO/FAO/UNU要求的全部必需氨基酸,蛋白质中的每一项必需氨基酸含量均高于WHO/FAO/UNU对成人(婴儿)的要求。
2.对利用低氟南极磷虾蛋白质基料制备蛋白粉进行了研究。结果表明,喷雾干燥制备蛋白粉时,部分前处理的适宜条件是控制进料干物质含量≥7.89%,调节物料至pH7.2,40Mp高压均质,进料液温度为60℃；制备的蛋白粉水分含量<5%,其总脂含量高、磷脂含量高和表面游离脂肪含量高；喷雾干燥过程中,较低的进料干物质含量导致液滴固化后颗粒表层蛋白质的含量较大,蛋白粉的变性程度增加,而进料干物质含量的增加导致液滴固化后脂质更易滞留在颗粒表面,蛋白质更倾向于被包埋在颗粒内部；制备的低氟南极磷虾蛋白粉在空气下容易氧化,充氮包装能够良好地抑制其氧化；充氮包装下,添加抗氧化剂的低氟南极磷虾蛋白粉的抗氧化性显著地(P<0.05)提高,其中,一种复合抗氧化剂(L一抗坏血酸棕榈酸酯+dl-α-醋酸生育酚,1+1)的效果最优。
3.低氟南极磷虾蛋白质基料制备过程中产生了两股废液副产物：酸沉废液和脱氟废液。本文研究了废液副产物的利用,即研究向废液副产物中添加南极磷虾,利用二段自溶技术制备高氨基酸态氮南极磷虾自溶液的效果。结果表明,酸沉废液和脱氟废液的含氮成分主要是小分子肽或氨基酸；在利用酸沉废液时,通过二段自溶技术制备的自溶液的氨基酸态氮达到1.070g/100mL,总氮达到1.97g/100mL,铵盐占氨基酸态氮的比例为21.18%,氟含量为18.85μg/mL,蛋白质利用率达到79.48%；在利用酸沉废液和脱氟废液的混合物时,通过二段自溶技术制备的自溶液的氨基酸态氮达到1.026g/100mL,总氮达到1.94g/100mL,铵盐占氨基酸态氮的比例为19.71%,氟含量为18.79μg/mL,蛋白质利用率达到77.32%。
4.对南极磷虾二段自溶技术进行了研究,即研究向南极磷虾中添加水,利用二段自溶技术制备高氨基酸态氮南极磷虾自溶液的效果。此外,研究了南极磷虾一段自溶的部分影响因素。
南极磷虾一段自溶影响因素的研究结果表明,合适浓度的Na+、K+、Ca2+、 Mg2+溶液对磷虾自溶均有显著的(P<0.05)促进作用,而Zn2+溶液对磷虾自溶的促进作用不显著(P>0.05),较高浓度的Zn2+溶液对磷虾自溶还具有显著的(P<0.05)抑制作用；适度的紫外线辐照预处理可使磷虾一段自溶液的氨基酸态氮含量达到0.739g/100mL。
南极磷虾二段自溶技术的研究结果表明,南极磷虾二段自溶时,第二段自溶的最优条件是：液料比2.23,初始pH7.24,温度40.9℃,时间12h。最优的二段自溶条件下制备的二段自溶液含有氨基酸态氮0.951g/100mL、总氮1.92g/100mL,这两者均高于国家标准《酿造酱油》GB18186-2000对低盐固态发酵酱油特级品的相应指标值要求；二段自溶液含有的铵盐占氨基酸态氮的比例符合国家标准《酿造酱油》(征求意见稿)GB18186的新规定,含有的氟为18.60μg/mL；二段自溶液的蛋白质利用率达到75.33%。南极磷虾二段自溶技术具有蛋白质利用率高、工序简单、设备投入小、生产周期短、自溶液的氨基酸态氮含量高的优点；制备的二段自溶液含有WHO/FAO/UNU要求的全部必需氨基酸,其中Trp、Thr、His和Phe+Tyr是限制性氨基酸。二段自溶技术大幅度提高了自溶液的鲜味氨基酸含量,二段自溶液的总鲜味氨基酸达到46.06mg/mL,比一段自溶液的总鲜味氨基酸提高了23.71mg/mL,提高率达到106.09%。
5.对南极磷虾二段自溶液的氟脱除进行了研究。结果表明,采用钙盐沉淀法对南极磷虾自溶液脱氟时,在氯化钙、乳酸钙和醋酸钙中,以乳酸钙的氟脱除效果最优；乳酸钙作用下的初步脱氟液含有氟8.19g/100mL,氨基酸态氮0.854g/100mL,总氮1.72g/100mL,其总氮损失率是9.54%；在应用乳酸钙脱氟的基础上,一种生物脱氟剂能够将南极磷虾自溶液的氟含量降低至1.53mg/L,从而解决了南极磷虾自溶液中氟的安全性问题；该生物脱氟剂作用下的深度脱氟液含有氟1.53g/100mL,氨基酸态氮0.783g/100mL,总氮1.67g/100mL,其总氮损失率是14.06%。
对南极磷虾二段自溶液的风味成分进行了研究。结果表明,2-甲基丁醛、3-甲基丁醛、3-甲基-1-丁醇、乙醇是南极磷虾自溶液的主体风味成分,其相对丰度分别为24.56%、17.79%、17.70%和5.14%；南极磷虾自溶液的总挥发性风味化合物中,2-甲基丁醛、3-甲基丁醛、3-甲基-1-丁醇这三种食品香料的总相对丰度高达60.05%。
The biomass of Antarctic krill is huge, and Antarctic krill are rich in proteins with high quality. But, the fluoride content of Antarctic krill is extremely high and Antarctic krill contains very strong proteolytic enzymes. Thus, based on these characters of Antarctic krill, the investigation of deep processing of Antarctic krill proteins is important to develop safty krill commodities wih high added value. Two sets of new technologies for deep processing of Antarctic krill proteins were investigated in this thesis. One was based on the preparation of proteins with low fluoride level from Antarctic krill, including the preparation of proteins with low fluoride level from Antarctic krill, the preparation of protein powders by using the proteins with low fluoride level, and the utilization of the byproducts of waste liquor produced during the preparation of proteins with low fluoride leve. Another was the preparation of hydrolysates with high amino acid nitrogen level by using the technology of two-stage krill autolysis, including the investigation of the technology of two-stage krill autolysis, and the investigation of the fluoride removal and volatile flavor compounds of hydrolysates obtained by two-stage krill autolysis. The main investigations and their results were as follows.
1. The alkali solubilization and acid precipitation method for recovering proteins from Antarctic krill was systematacially investigated. Based on the investigation of the method, the fluoride removal technology of the acid-precipitated proteins obtained by the method was investigated, and the proteins with low fluoride level were prepared. Besides, the composition of the proteins with low fluoride level was analyzed. The optimal conditions for protein solubilization were determined to be pH11.5and4℃. The optimum method of protein solubilization was the proteins were solubilized two times; a water/krill ratio (mL/g) of6and a time of30min were used for the first step, whereas the second used a water/krill residue ratio (mL/g) of3and a time of30min. The dissolved protein rate by the optimum method of protein solubilization was97.23%. The optimum pH for protein precipitation was4.6. The protein recovery yield was increased by5%when transglutaminase was correctly applied during the acid-induced protein precipitation. The proteins with low fluoride level were finally obtained through an effective fluoride removal technology of the acid-precipitated proteins. The fluoride content of the proteins with low fluoride level was lower than2mg/kg. The protein yield of the proteins with low fluoride level was52.68%. The proteins with low fluoride level were composed of66.96%of crude proteins (dry weight) and33.01%of total lipids (dry weight). The fatty acids of the proteins with low fluoride level were rich in EPA(13.65%)、DHA(7.17%)和MUFAs (16:18.70%,18:120.61%). The proteins with low fluoride level contained all essential amino acids in sufficient amounts to meet WHO/FAO/UNU requirements for adults and infants.
2. The preparation of protein powders by using the proteins with low fluoride level was investigated. The optimal conditions of some pretreatments of spray drying were determined as follows:the dry matter content of feeds (≥7.89%), the adjusted pH of materials (7.2), the high pressure of homogenization (40Mp), the temperature of feeds (60℃). The moisture content of the prepared protein powders was lower than5%, and the protein powders contained high levels of total lipids, total phospholipids and surface free-fat. During the spray drying process, a lower dry matter content of feeds resulted in a higher protein content on the particle surface after the liquid drops were solidified, which made the denaturation degree of protein powders increase, whereas an increased dry matter content of feeds led to more lipids existing on the particle surface and more proteins enclosed in the particle interior after the liquid drops were solidified. The prepared protein powders with low fluoride level exhibited a bad oxidation resistance when they were exposed to air, and their easy oxidation properties were well inhibited by nitrogen-filled packages. Under the condition of nitrogen-filled packages, the antioxidant properties of the protein powders with antioxidant addition were significantly (P<0.05) improved, and a combined antioxidant (L-ascorbyl palmitate+DL-a-tocopheryl acetate,1+1) exhibited the best antioxidant performance.
3. The preparation of the proteins with low fluoride level resulted in two waste liquor byproducts, of which one was produced during the acid-induced protein precipitation, and another was produced during the fluoride removal of the acid-precipitated proteins. The utilization of the byproducts was investigated through the addition of Antarctic krill to the byproducts, followed by the preparation of hydrolysates with high amino acid nitrogen level using the technology of two-stage krill autolysis in this thesis. The main nitrogen-containing ingredients of waste liquor byproducts were small molecules of peptides or amino acids. Through the utilization of waste liquor produced during the acid-induced protein precipitation, the hydrolysates obtained by two-stage krill autolysis contained1.070g/100mL of amino acid nitrogen,1.97g/100mL of total nitrogen,18.85μg/mL of fluoride, meanwhile the ammonium salt to amino acid nitrogen rate and protein yield of the hydrolysates were21.18%and79.48%, respectively. Through the utilization of the mixture of waste liquor produced during the acid-induced protein precipitation and the fluoride removal of the acid-precipitated proteins, the hydrolysates obtained by two-stage krill autolysis contained1.026g/100mL of amino acid nitrogen,1.94g/100mL of total nitrogen,18.79μg/mL of fluoride, meanwhile the ammonium salt to amino acid nitrogen rate and protein yield of the hydrolysates were19.71%and77.32%, respectively.
4. The technology of two-stage krill autolysis was investigated. During the investigation, distilled water was added to Antarctic krill, and the preparation of hydrolysates with high amino acid nitrogen level using the technology was investigated. Furthermore, the partial influence factors of one-stage krill autolysis were investigated.
The investigation of the influence factors of one-stage krill autolysis indicated Na+, K+, Ca2+and Mg2+solutions at appropriate concentrations showed significantly (P<0.05) promoting effects on krill autolysis, respectively, and the promoting effect of Zn2+solution on krill autolysis was insignificant (P>0.05). Furthermore, the Zn2+solution at high concentration exhibited significantly (P<0.05) inhibitory effects on krill autolysis. The amino acid nitrogen content of hydrolysates obtained by one-stage krill autolysis was0.739g/100mL through the pretreatment of appropriate ultraviolet radiation.
The technology of two-stage krill autolysis was investigated. The optimal conditions of the second-stage krill autolysis during two-stage krill autolysis were determined to be a liquor to material rate of2.23(g/g), an initial pH of7.24, a temperature of40.9℃, a time of12h. Under these optimal conditions, the hydrolysates obtained by two-stage krill autolysis contained1.026g/100mL of amino acid nitrogen and1.92g/100mL of total nitrogen, and each of them was in sufficient amount to meet Chinese National Standard GB18186-2000requirements for the special grade soy sauce of low salt-solid fermentation. The ammonium salt to amino acid nitrogen rate of the hydrolysates met the new requirement of Chinese National Standard GB18186(Draft for Comment) for fermented soy sauce, and the fluoride content and protein yield of the hydrolysates were18.60μg/mL and75.33%, respectively. The technology of two-stage krill autolysis had the advantages of high protein yield, simple treatment processes, low equipment investment, short processing time, and prepared hydrolysates with high amino acid nitrogen level. The prepared hydrolysates contained all essential amino acids required by WHO/FAO/UNU, of which Trp, Thr, His and Phe+Tyr were the limiting amino acids. The technology of two-stage krill autolysis could greatly improve the total flavour amino acid content of the hydrolysates, which was46.06mg/mL. The total flavour amino acid content of hydrolysates obtained by two-stage krill autolysis increased by23.71mg/mL compared to that of hydrolysates obtained by one-stage krill autolysis, and the increasing rate was106.09%.
5. The fluoride removal of hydrolysates obtained by two-stage krill autolysis was investigated. Compared to calcium chloride and calcium acetate, calcium lactate exhibited the best fluoride removal efficiency when the calcium precipitation method was employed to remove fluoride from the hydrolysates. The solution obtained by the preliminary fluoride removal of the hydrolysates through the application of calcium lactate contained8.19μg/mL of fluoride,0.854g/100mL of amino acid nitrogen,1.72 g/100mL of total nitrogen, and the loss rate of total nitrogen was9.54%. Baed on the preliminary fluoride removal of the hydrolysates through the application of calcium lactate, a biological defluorination agent could efficiently remove the residuaful fluoride from the hydrolysates and make the fluoride content of the hydrolysates decrease to1.53μg/100mL. Therefor, the safty problem of fluoride in the hydrolysates was successfully resolved. The solution obtained by the deep fluoride removal of the hydrolysates through the application of the biological defluorination agent contained1.53μg/mL of fluoride,0.783g/100mL of amino acid nitrogen,1.67g/100mL of total nitrogen, and the loss rate of total nitrogen was14.06%.
The volatile flavor compounds of hydrolysates obtained by two-stage krill autolysis were investigated. The main volatile compounds of the hydrolysates were identified as2-methyl-Butanal,3-methyl-Butanal,3-methyl-1-Butanol, Ethanol, and each of their relative abundance in the identified volatiles was24.56%,17.79%,17.70%and5.14%, respectively. The three compounds of2-methyl-Butanal,3-methyl-Butanal and3-methyl-1-Butanol are permited to use as food flavors, and their total relative abundance was60.05%.
引文
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