竹取物在食品品质改良及风险控制中的应用基础研究
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摘要
我国素有“竹子王国”之称,竹类资源丰富,竹林面积广阔,竹文化源远流长,来自竹林的众多产品具有悠久的食用和药用历史。竹取物(Extract of Bamboo)是指运用中药现代化技术和手段,从竹子不同部位获得的具有多种生理和药理活性的植物次生代谢产物(又称竹子化学素),包括竹叶黄酮、竹叶抗氧化物、竹茹三萜、竹笋甾醇、竹笋ACE抑制肽、竹子多糖等等。
竹叶抗氧化物(Antioxidant of Bamboo Leaves, AOB),取自刚竹属(Phyllostachys Sieb.et Zucc.)竹叶的酚性部位,于2004年被批准列入《中华人民共和国食品添加剂使用卫生标准》(GB2760-2011)。有效成分主要为黄酮和酚酸类化合物,除了具有良好的抗汕脂氧化性能外,同时具有卓越的抑制热加工食品中内源性化学污染物(丙烯酰胺,俗称丙毒)形成的能力。
竹青素(Green Bamboo Extract, GBE),以刚竹属竹子为原料,采用专利技术萃取竹子不同部分的有效成分,集竹叶多酚、竹茹多糖和竹笋多肽于一体,是一种生物抗氧化剂、脂代谢调节剂和免疫功能增强剂,内含丰富的植物次生代谢产物,主要包括黄酮、酚酸、多糖、三萜、内酯、蒽醌、氨基酸、多肽以及矿质元素等,具有作为天然物饲料添加剂研究开发和牛产应用的巨大潜力。
本学位论文重点研究：1)AOB同时作为食品抗氧化剂和丙烯酰胺抑制剂在曲奇(种高油、高糖、高蛋白的酥性饼十)加工中的应用；2)GBE作为天然物饲料添加剂对养殖大黄鱼食用品质的改善和食用安全性的提高,以期为竹取物在食品和饲料行业的应用提供理论依据和实践指导。现将主要研究成果归纳如下：
1、总黄酮含量测定是竹取物制剂质量控制的关键点之一。硝酸铝-亚硝酸钠可见分光光度法是目前用于测定植物提取物及其制品中总黄酮含量的最常用方法,通常以芦丁作为标准品,在510nm波长下进行测定。由于比色分析的十扰因素多,测定误差大,同时,竹叶及其提取物中基本上不含芦丁,故采用传统的总黄酮测定方法(硝酸铝-亚硝酸钠可见分光光度法)来标化竹叶提取物的质量存在一定的缺陷。本文首次建立了以异荭草苷(竹叶中最主要的一种黄酮类化合物)为标准品的紫外分光光度法,并与传统方法进行比较。利用聚酰胺吸附-解析净化处理竹叶提取物试样后,选择在340nm的紫外波长下、以异荭草苷作为标准品直接进行测定。方法学论证显示,该方法简捷、准确、灵敏度高、结果可靠,比传统硝酸铝-亚硝酸钠可见分光光度法更适用于竹叶提取物及其相关产品中总黄酮含量的测定。
2、利用正交试验优化了微波提取竹叶总黄酮的工艺参数。通过极差分析确定了对黄酮提取率影响较大的因素依次为：乙醇浓度>微波功率>料液比>微波处理时间；方差分析显示,乙醇浓度、料液比和微波功率对黄酮提取率的影响极显著(p<0.01),微波处理时间的影响显著(p<0.05)。采用优化后的工艺参数(提取液乙醇浓度12.5%,料液比1：12,微波处理时间4.5mmin,微波功率900W),竹叶总黄酮的提取率为83.23%。研究表明,对比传统的热回流工艺,微波提取竹叶黄酮的效率明显提升,溶剂消耗少,加热时间短,有利于节能降耗、保护环境、提高效益,为规模化生产提供技术支持。
3、将食品添加剂AOB应用于曲奇加工,可有效降低制品中丙烯酰胺的含量。结合体外抗氧化体系(DPPH和ABTS体系)评价发现,不同加工方法和制剂精度的AOB的抗氧化活性与其总黄酮含量成显著正相关。采用大孔树脂吸附-解析得到的AOB制剂,虽然总黄酮含量和抗氧化活性都高于采用有机溶剂液-液萃取精制得到的AOB制剂,但其对曲奇中丙稀酰胺形成的抑制效果却明显低于后者。研究发现,热加工食品中丙烯酰胺的抑制效果并非单纯依赖于AOB制剂的总黄酮含量,也不能仅用抗氧化能力的强弱来推断,AOB中所含的其他有效成分物质(如酚酸等)也是丙烯酰胺抑制的重要因素。用DPPH法,对曲奇进行抗氧化性能测定,发现添加AOB抑制丙烯酰胺形成的同时,产品体系的抗氧化性也随之降低,表明AOB在一定程度上对美拉德反应产生了影响,可能导致具有抗氧化活性的类黑精物质形成减少。研究比较了五种不同的食.品品抗氧化剂对曲奇丙烯酰胺形成的抑制效果,同时结合产品的感官评定,发现配料中添加0.2g/kg的AOB或0.1g/kg VE的均能取得理想效果,抑制率分别可达63.9%和71.2%,且能有效保持曲奇的感官品质。表明,AOB作为抗氧化和丙毒抑制的多功能食品添加剂在焙烤食品中具有广阔的应用前景。
4、海水网箱养殖试验表明,将GBE应用于大黄鱼的人工养殖,可激活大黄鱼的脂质代谢,增强鱼体免疫力和抗应激能力,显著改善品质,并提高食用安全性。采用添加0.3%GBE的新型生态环保饲料(大黄鱼圈价膨化颗粒饲料,记作0#料)饲喂大黄鱼,与传统冰鲜喂养的大黄鱼及野生大黄鱼进行比较分析。饲喂6个月后,试验组大黄鱼的肝脏、腹肌、背肌中的脂肪含量均较冰鲜对照组显著减少,肝体比降低,脂肪肝症状缓解；鱼体脂肪酸组成中多不饱和脂肪酸(特别是亚汕酸、亚麻酸)含量增加。感官评价试验表明,试验组鱼品在风味、肉质、口感上与野生大黄鱼相似,相比传统冰鲜喂养的大黄鱼,品质大幅度提高。电镜观察表明,试验组大黄鱼肌肉中脂肪明显减少,胶原和线粒体丰富,肌丝条纹更接近野生大黄鱼。并且,用强化GBE的全价饲料代替冰鲜喂养,大大提高了养殖对象的免疫能力,增强了抗病性。同时,分析检测还表明,添加GBE的试验组成鱼体内积累的化学污染物(二噁英、多氯联苯和多溴联苯)的含量相比野生大黄鱼有所降低,食用安全性提高。
5、在上述探索性试验的基础上,进一步优化GBE中来自竹子不同部位的植物化学素(竹叶提取物、竹茹提取物和竹笋提取物)的配比,加工成三种GBE添加量均为0.3%的大黄鱼全价膨化颗粒饲料,分别记作1“料、2“料和3#料,同时设置0.1%虾青素含量的阳性对照组、传统冰鲜喂养对照组及原GBE配方的0#料组,同步进行标准海水网箱的养殖对比试验。结果表明,1#和2“料在降低肌肉脂肪和肝脏脂肪含量方面均优于0#料,综合养殖效果接近甚至优于虾青素阳性对照。检测大黄鱼肝脏中脂联素的水平,表明虾青素组、1“料和2#料组饲养的大黄鱼肝脏脂联素水平升高,表明脂质代谢能力增强。同时,强化GBE的大黄鱼全价配合饲料能有效提升大黄鱼体内抗氧化酶(CAT和SOD)的活性,并显著降低脂质过氧化产物(MDA)的水平,减轻肝脏损伤。感官评价试验证明,1#和2“试验组鱼肉的风味和口感极大地超越了传统冰鲜喂养的大黄鱼。鱼肉氨基酸组成分析结果显示,食用品质的提升与鱼肉中呈味氨基酸总量的增加有关。综合分析表明,含0.3%GBE2#试验料在增加大黄鱼的氨基酸总量、必需氨基酸和呈味氨基酸含量、多不饱和脂肪酸(尤其是DHA和EPA)含量以及UFA/SFA指数等方面的作用可与添加0.1%虾青素的阳性对照媲美。表明GBE作为一种天然物饲料添加剂在高端水产品养殖业中具有广阔的应用前景。
China is known as'the kingdom of bamboo'. It is rich in not only the area of bamboo forest, but also the bamboo culture with long history. There are a wide range of edible and medicinal products from bamboo that have been used for a long history. Bamboo extract is a series of plant metabolites, produced from different parts of bamboo via modern technology, including flavone of bamboo leaves, antioxidant of bamboo leaves, triterpenoid of bamboo shavings, sterol of bamboo shoot, peptide of bamboo shoot, bamboo polysaccharide, etc.
Antioxidant of bamboo leaves (AOB), is produced from the phenol composition of Phyllostachys Sieb. Et Zucc., in which the flavone of bamboo leaves is the main ingredient, and also approved as food antioxidants in Chinese national standards (GB-2760). AOB, with the flavonoids and phenol acids as main compounds, could not only inhibit oil oxidation, but also mitigate the formation of endogenous chemical pollutants (acrylamide) in the heating food producing.
Green bamboo extract (GBE), extracted from different parts of bamboo by patent technology, including the phenol acids of bamboo leaves, polysaccharide of bamboo shavings and peptide of bamboo shoot. It is a kind of excellent biological antioxidants, lipid metabolism regulator and immune function reinforcing agent, rich in plant secondary metabolites, which mainly included flavones, phenolic acid, polysaccharide, terpene lactones, anthraquinone, amino acids, polypeptides, mineral element and so on. GBE has the huge potential of research and application as feed additive.
The key research contents:1) AOB was used in cookie (a kind of crisp biscuit with high level of oil, sugar, protein) processing as a food antioxidant and acrylamide inhibitor;2) GBE was used in cultured large yellow croaker as a feed additive to improve the edible quality and safety. The main research results were summarized as follows:
1) Determination of total flavonoid content is the key point of bamboo extract quality control. Researchers found that bamboo extract contains a considerable amount of poly-hydroxyl phenolic acid, which also could chelate with aluminium ion, affecting the chromogenic reaction results; In addition, bamboo extract contains no rutin, but isoorientin as its characteristic flavonoid. These two reasons made the Al (NO3)3-NaNO2colorimetric method using rutin as standard not suitable for the total flavonoids determination of bamboo extract. This research established the ultraviolet spectrophotometric method with isoorientin as the standard in detection of total flavonoids of bamboo extract for the first time. After polyamide purification, bamboo extract could be directly detected at340nm with isoorientin as the standard, befitting the rapid detection in the bamboo extract production. This method was more suitable for the total flavonoids determination of bamboo extract and subsequent products than the Al (NO3)3-NaNO2colorimetric method, for its high reliability, accuracy and sensitivity.
2) We optimized the microwave extraction technology of total flavonoids in bamboo leaves by orthogonal experiment. The influence of four factors on total flavonoids yield was sequenced by range analysis as follows:ethanol concentration> microwave power> liquid-solid ratio> microwave irradiation time. In the variance analysis, the ethanol concentration, microwave power and liquid-solid ratio affected the flavonoid yield very significantly (p<0.0\), while microwave irradiation time performed significantly (p<0.05). In the condition of ethanol concentration (12.5%), solid-liquid ratio (1:12), microwave irradiation time (4.5min) and microwave power (900W), the flavonoid yield could reach83.23%. Compared to the traditional heat reflux extraction, microwave technology could be more effective in the flavonoid extraction of bamboo leaves, with lower consumption of organic solvent, less heating time. This technology was an environmentally friendly and low-energy consumesd method, which would be more suitable for the large-scale production of bamboo leaves extract.
3) Antioxidant of bamboo leaves (AOB), a legal food additive, used in cookies processing, could reduce the acrylamide level effectively. Tested in the antioxidant evaluation in-vitro system, antioxidant activity of AOB was found positively correlated with the content of flavonoid. The AOB prepared by solution adsorption method performed better than the AOB purificated by macro porous resin in acrylamide inhibition, although the latter had higher flavonoid content and stronger antioxidant activity. According to the composition analysis, the acrylamide inhibition ability of food additive couldn't be supposed to relate with flavonoid content and the antioxidant activity, which maybe actually relate with the other substances (such as phenolic acid, etc.) in the antioxidant. The antioxidant activity of cookie system decreased, along with the acrylamide inhibition by exogenous AOB. It indicated that AOB could influence the Millard reaction processing at a certain degree, leading to the reduction of melanoidins, which contributed obviously to the antioxidant activity. In comparison of five antioxidants in the inhibition of acrylamide formation and sensory evaluation, AOB (0.2g/kg) and VE (0.1g/kg) mitigated the formation of acrylamide by63.9%and71.2%, respectively, with the achievement of the balance between acrylamide reduction and sensory acceptance. This work showed the potential effectiveness of AOB in practical food system for decreasing acrylamide formation.
4) Green bamboo extract (GBE) was used in the cultivation of large yellow croaker, to activate its lipid metabolism, strengthen the body immunity and anti-stress ability, improve the quality and ameliorate the edible safety. The large yellow croaker fed by the new environmental feed added with0.3%green bamboo extract (0#feed), was compared with the control group and the wild ones. The results indicated that there is significant decrease in the fat content in the liver, abdominal muscle and back muscle of test group, after6-month feeding. The fatty liver disease symptom was improved obviously, along with the reduction of liver-body ratio. Moreover, the polyunsaturated fatty acids (particularly linoleic acid and linolenic acid) level in the fish meat increased, which was very important to the nutrition value and fish immunity. Sensory evaluation, including the flavor, meat quality and taste, showed that the similarity between0#group and wild ones was close to80%, with an obvious sensory improvement compared to control group. Transmission electron microscope results showed that the fish muscle from test group, whose muscle texture was closer to the wild group, was rich in collagen and mitochondria, but low in fat. In addition, large yellow croakers fed by GBE had built up greater immunity, and stronger disease resistance. In the measurement by a gas chromatograph coupled to a high resolution mass spectrometer, persistent organic pollutants level (including polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated biphenyls, and polybrominated diphenyl ethers) in fish from GBE group was lower than in wild group, which could be considered as a key factor to the edible safety.
5) Based on the previous experiments, GBE was optimized by adjusting the ratio of bamboo leaves extract, bamboo shavings extract and bamboo shoots extract as three formulas(1#,2#,3#), which was then added in the feed by0.3%, compared with control group,0.1%astaxanthin positive control group and0#group in the sea-cages breeding of large yellow croaker. The results showed that1#and2#formulas performed better in reducing muscle fat and liver fat content than0#, close to and even better than the effect of astaxanthin. In measurement of adiponectin level, fish from1#and2#group activated in lipid metabolism, for the higher level of adiponectin, which indicated that the way of promoting lipid metabolism by these formulas was via regulating lipid metabolism related hormone. Feed added by GBE could effectively regulated the CAT and SOD activity, which could be considered as an important factor to enhance the body against free radicals, especially the degradation products of polyunsaturated fatty acid peroxidation (MDA, etc.), avoiding the liver damage. It was possible that the composition of flavour amino acids was the major cause of sensory improvement of GBE groups, according to the results of sensory evaluation and amino acid determination. Comprehensively, the feed added with0.3%GBE2#could increase the level of total amino acids, essential amino acids, flavour amino acids, polyunsaturated fatty acid (especially DHA and EPA) and UFA/SFA index, etc. similar to the effect of0.1%astaxanthin. It showed that GBE could be used as a natural feed additive in high-end aquatic breeding.
竹叶抗氧化物(Antioxidant of Bamboo Leaves, AOB),取自刚竹属(Phyllostachys Sieb.et Zucc.)竹叶的酚性部位,于2004年被批准列入《中华人民共和国食品添加剂使用卫生标准》(GB2760-2011)。有效成分主要为黄酮和酚酸类化合物,除了具有良好的抗汕脂氧化性能外,同时具有卓越的抑制热加工食品中内源性化学污染物(丙烯酰胺,俗称丙毒)形成的能力。
竹青素(Green Bamboo Extract, GBE),以刚竹属竹子为原料,采用专利技术萃取竹子不同部分的有效成分,集竹叶多酚、竹茹多糖和竹笋多肽于一体,是一种生物抗氧化剂、脂代谢调节剂和免疫功能增强剂,内含丰富的植物次生代谢产物,主要包括黄酮、酚酸、多糖、三萜、内酯、蒽醌、氨基酸、多肽以及矿质元素等,具有作为天然物饲料添加剂研究开发和牛产应用的巨大潜力。
本学位论文重点研究：1)AOB同时作为食品抗氧化剂和丙烯酰胺抑制剂在曲奇(种高油、高糖、高蛋白的酥性饼十)加工中的应用；2)GBE作为天然物饲料添加剂对养殖大黄鱼食用品质的改善和食用安全性的提高,以期为竹取物在食品和饲料行业的应用提供理论依据和实践指导。现将主要研究成果归纳如下：
1、总黄酮含量测定是竹取物制剂质量控制的关键点之一。硝酸铝-亚硝酸钠可见分光光度法是目前用于测定植物提取物及其制品中总黄酮含量的最常用方法,通常以芦丁作为标准品,在510nm波长下进行测定。由于比色分析的十扰因素多,测定误差大,同时,竹叶及其提取物中基本上不含芦丁,故采用传统的总黄酮测定方法(硝酸铝-亚硝酸钠可见分光光度法)来标化竹叶提取物的质量存在一定的缺陷。本文首次建立了以异荭草苷(竹叶中最主要的一种黄酮类化合物)为标准品的紫外分光光度法,并与传统方法进行比较。利用聚酰胺吸附-解析净化处理竹叶提取物试样后,选择在340nm的紫外波长下、以异荭草苷作为标准品直接进行测定。方法学论证显示,该方法简捷、准确、灵敏度高、结果可靠,比传统硝酸铝-亚硝酸钠可见分光光度法更适用于竹叶提取物及其相关产品中总黄酮含量的测定。
2、利用正交试验优化了微波提取竹叶总黄酮的工艺参数。通过极差分析确定了对黄酮提取率影响较大的因素依次为：乙醇浓度>微波功率>料液比>微波处理时间；方差分析显示,乙醇浓度、料液比和微波功率对黄酮提取率的影响极显著(p<0.01),微波处理时间的影响显著(p<0.05)。采用优化后的工艺参数(提取液乙醇浓度12.5%,料液比1：12,微波处理时间4.5mmin,微波功率900W),竹叶总黄酮的提取率为83.23%。研究表明,对比传统的热回流工艺,微波提取竹叶黄酮的效率明显提升,溶剂消耗少,加热时间短,有利于节能降耗、保护环境、提高效益,为规模化生产提供技术支持。
3、将食品添加剂AOB应用于曲奇加工,可有效降低制品中丙烯酰胺的含量。结合体外抗氧化体系(DPPH和ABTS体系)评价发现,不同加工方法和制剂精度的AOB的抗氧化活性与其总黄酮含量成显著正相关。采用大孔树脂吸附-解析得到的AOB制剂,虽然总黄酮含量和抗氧化活性都高于采用有机溶剂液-液萃取精制得到的AOB制剂,但其对曲奇中丙稀酰胺形成的抑制效果却明显低于后者。研究发现,热加工食品中丙烯酰胺的抑制效果并非单纯依赖于AOB制剂的总黄酮含量,也不能仅用抗氧化能力的强弱来推断,AOB中所含的其他有效成分物质(如酚酸等)也是丙烯酰胺抑制的重要因素。用DPPH法,对曲奇进行抗氧化性能测定,发现添加AOB抑制丙烯酰胺形成的同时,产品体系的抗氧化性也随之降低,表明AOB在一定程度上对美拉德反应产生了影响,可能导致具有抗氧化活性的类黑精物质形成减少。研究比较了五种不同的食.品品抗氧化剂对曲奇丙烯酰胺形成的抑制效果,同时结合产品的感官评定,发现配料中添加0.2g/kg的AOB或0.1g/kg VE的均能取得理想效果,抑制率分别可达63.9%和71.2%,且能有效保持曲奇的感官品质。表明,AOB作为抗氧化和丙毒抑制的多功能食品添加剂在焙烤食品中具有广阔的应用前景。
4、海水网箱养殖试验表明,将GBE应用于大黄鱼的人工养殖,可激活大黄鱼的脂质代谢,增强鱼体免疫力和抗应激能力,显著改善品质,并提高食用安全性。采用添加0.3%GBE的新型生态环保饲料(大黄鱼圈价膨化颗粒饲料,记作0#料)饲喂大黄鱼,与传统冰鲜喂养的大黄鱼及野生大黄鱼进行比较分析。饲喂6个月后,试验组大黄鱼的肝脏、腹肌、背肌中的脂肪含量均较冰鲜对照组显著减少,肝体比降低,脂肪肝症状缓解；鱼体脂肪酸组成中多不饱和脂肪酸(特别是亚汕酸、亚麻酸)含量增加。感官评价试验表明,试验组鱼品在风味、肉质、口感上与野生大黄鱼相似,相比传统冰鲜喂养的大黄鱼,品质大幅度提高。电镜观察表明,试验组大黄鱼肌肉中脂肪明显减少,胶原和线粒体丰富,肌丝条纹更接近野生大黄鱼。并且,用强化GBE的全价饲料代替冰鲜喂养,大大提高了养殖对象的免疫能力,增强了抗病性。同时,分析检测还表明,添加GBE的试验组成鱼体内积累的化学污染物(二噁英、多氯联苯和多溴联苯)的含量相比野生大黄鱼有所降低,食用安全性提高。
5、在上述探索性试验的基础上,进一步优化GBE中来自竹子不同部位的植物化学素(竹叶提取物、竹茹提取物和竹笋提取物)的配比,加工成三种GBE添加量均为0.3%的大黄鱼全价膨化颗粒饲料,分别记作1“料、2“料和3#料,同时设置0.1%虾青素含量的阳性对照组、传统冰鲜喂养对照组及原GBE配方的0#料组,同步进行标准海水网箱的养殖对比试验。结果表明,1#和2“料在降低肌肉脂肪和肝脏脂肪含量方面均优于0#料,综合养殖效果接近甚至优于虾青素阳性对照。检测大黄鱼肝脏中脂联素的水平,表明虾青素组、1“料和2#料组饲养的大黄鱼肝脏脂联素水平升高,表明脂质代谢能力增强。同时,强化GBE的大黄鱼全价配合饲料能有效提升大黄鱼体内抗氧化酶(CAT和SOD)的活性,并显著降低脂质过氧化产物(MDA)的水平,减轻肝脏损伤。感官评价试验证明,1#和2“试验组鱼肉的风味和口感极大地超越了传统冰鲜喂养的大黄鱼。鱼肉氨基酸组成分析结果显示,食用品质的提升与鱼肉中呈味氨基酸总量的增加有关。综合分析表明,含0.3%GBE2#试验料在增加大黄鱼的氨基酸总量、必需氨基酸和呈味氨基酸含量、多不饱和脂肪酸(尤其是DHA和EPA)含量以及UFA/SFA指数等方面的作用可与添加0.1%虾青素的阳性对照媲美。表明GBE作为一种天然物饲料添加剂在高端水产品养殖业中具有广阔的应用前景。
China is known as'the kingdom of bamboo'. It is rich in not only the area of bamboo forest, but also the bamboo culture with long history. There are a wide range of edible and medicinal products from bamboo that have been used for a long history. Bamboo extract is a series of plant metabolites, produced from different parts of bamboo via modern technology, including flavone of bamboo leaves, antioxidant of bamboo leaves, triterpenoid of bamboo shavings, sterol of bamboo shoot, peptide of bamboo shoot, bamboo polysaccharide, etc.
Antioxidant of bamboo leaves (AOB), is produced from the phenol composition of Phyllostachys Sieb. Et Zucc., in which the flavone of bamboo leaves is the main ingredient, and also approved as food antioxidants in Chinese national standards (GB-2760). AOB, with the flavonoids and phenol acids as main compounds, could not only inhibit oil oxidation, but also mitigate the formation of endogenous chemical pollutants (acrylamide) in the heating food producing.
Green bamboo extract (GBE), extracted from different parts of bamboo by patent technology, including the phenol acids of bamboo leaves, polysaccharide of bamboo shavings and peptide of bamboo shoot. It is a kind of excellent biological antioxidants, lipid metabolism regulator and immune function reinforcing agent, rich in plant secondary metabolites, which mainly included flavones, phenolic acid, polysaccharide, terpene lactones, anthraquinone, amino acids, polypeptides, mineral element and so on. GBE has the huge potential of research and application as feed additive.
The key research contents:1) AOB was used in cookie (a kind of crisp biscuit with high level of oil, sugar, protein) processing as a food antioxidant and acrylamide inhibitor;2) GBE was used in cultured large yellow croaker as a feed additive to improve the edible quality and safety. The main research results were summarized as follows:
1) Determination of total flavonoid content is the key point of bamboo extract quality control. Researchers found that bamboo extract contains a considerable amount of poly-hydroxyl phenolic acid, which also could chelate with aluminium ion, affecting the chromogenic reaction results; In addition, bamboo extract contains no rutin, but isoorientin as its characteristic flavonoid. These two reasons made the Al (NO3)3-NaNO2colorimetric method using rutin as standard not suitable for the total flavonoids determination of bamboo extract. This research established the ultraviolet spectrophotometric method with isoorientin as the standard in detection of total flavonoids of bamboo extract for the first time. After polyamide purification, bamboo extract could be directly detected at340nm with isoorientin as the standard, befitting the rapid detection in the bamboo extract production. This method was more suitable for the total flavonoids determination of bamboo extract and subsequent products than the Al (NO3)3-NaNO2colorimetric method, for its high reliability, accuracy and sensitivity.
2) We optimized the microwave extraction technology of total flavonoids in bamboo leaves by orthogonal experiment. The influence of four factors on total flavonoids yield was sequenced by range analysis as follows:ethanol concentration> microwave power> liquid-solid ratio> microwave irradiation time. In the variance analysis, the ethanol concentration, microwave power and liquid-solid ratio affected the flavonoid yield very significantly (p<0.0\), while microwave irradiation time performed significantly (p<0.05). In the condition of ethanol concentration (12.5%), solid-liquid ratio (1:12), microwave irradiation time (4.5min) and microwave power (900W), the flavonoid yield could reach83.23%. Compared to the traditional heat reflux extraction, microwave technology could be more effective in the flavonoid extraction of bamboo leaves, with lower consumption of organic solvent, less heating time. This technology was an environmentally friendly and low-energy consumesd method, which would be more suitable for the large-scale production of bamboo leaves extract.
3) Antioxidant of bamboo leaves (AOB), a legal food additive, used in cookies processing, could reduce the acrylamide level effectively. Tested in the antioxidant evaluation in-vitro system, antioxidant activity of AOB was found positively correlated with the content of flavonoid. The AOB prepared by solution adsorption method performed better than the AOB purificated by macro porous resin in acrylamide inhibition, although the latter had higher flavonoid content and stronger antioxidant activity. According to the composition analysis, the acrylamide inhibition ability of food additive couldn't be supposed to relate with flavonoid content and the antioxidant activity, which maybe actually relate with the other substances (such as phenolic acid, etc.) in the antioxidant. The antioxidant activity of cookie system decreased, along with the acrylamide inhibition by exogenous AOB. It indicated that AOB could influence the Millard reaction processing at a certain degree, leading to the reduction of melanoidins, which contributed obviously to the antioxidant activity. In comparison of five antioxidants in the inhibition of acrylamide formation and sensory evaluation, AOB (0.2g/kg) and VE (0.1g/kg) mitigated the formation of acrylamide by63.9%and71.2%, respectively, with the achievement of the balance between acrylamide reduction and sensory acceptance. This work showed the potential effectiveness of AOB in practical food system for decreasing acrylamide formation.
4) Green bamboo extract (GBE) was used in the cultivation of large yellow croaker, to activate its lipid metabolism, strengthen the body immunity and anti-stress ability, improve the quality and ameliorate the edible safety. The large yellow croaker fed by the new environmental feed added with0.3%green bamboo extract (0#feed), was compared with the control group and the wild ones. The results indicated that there is significant decrease in the fat content in the liver, abdominal muscle and back muscle of test group, after6-month feeding. The fatty liver disease symptom was improved obviously, along with the reduction of liver-body ratio. Moreover, the polyunsaturated fatty acids (particularly linoleic acid and linolenic acid) level in the fish meat increased, which was very important to the nutrition value and fish immunity. Sensory evaluation, including the flavor, meat quality and taste, showed that the similarity between0#group and wild ones was close to80%, with an obvious sensory improvement compared to control group. Transmission electron microscope results showed that the fish muscle from test group, whose muscle texture was closer to the wild group, was rich in collagen and mitochondria, but low in fat. In addition, large yellow croakers fed by GBE had built up greater immunity, and stronger disease resistance. In the measurement by a gas chromatograph coupled to a high resolution mass spectrometer, persistent organic pollutants level (including polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated biphenyls, and polybrominated diphenyl ethers) in fish from GBE group was lower than in wild group, which could be considered as a key factor to the edible safety.
5) Based on the previous experiments, GBE was optimized by adjusting the ratio of bamboo leaves extract, bamboo shavings extract and bamboo shoots extract as three formulas(1#,2#,3#), which was then added in the feed by0.3%, compared with control group,0.1%astaxanthin positive control group and0#group in the sea-cages breeding of large yellow croaker. The results showed that1#and2#formulas performed better in reducing muscle fat and liver fat content than0#, close to and even better than the effect of astaxanthin. In measurement of adiponectin level, fish from1#and2#group activated in lipid metabolism, for the higher level of adiponectin, which indicated that the way of promoting lipid metabolism by these formulas was via regulating lipid metabolism related hormone. Feed added by GBE could effectively regulated the CAT and SOD activity, which could be considered as an important factor to enhance the body against free radicals, especially the degradation products of polyunsaturated fatty acid peroxidation (MDA, etc.), avoiding the liver damage. It was possible that the composition of flavour amino acids was the major cause of sensory improvement of GBE groups, according to the results of sensory evaluation and amino acid determination. Comprehensively, the feed added with0.3%GBE2#could increase the level of total amino acids, essential amino acids, flavour amino acids, polyunsaturated fatty acid (especially DHA and EPA) and UFA/SFA index, etc. similar to the effect of0.1%astaxanthin. It showed that GBE could be used as a natural feed additive in high-end aquatic breeding.
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