CN117701478B - 一株高产果胶酶的嗜气芽孢杆菌及其应用 - Google Patents
一株高产果胶酶的嗜气芽孢杆菌及其应用 Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23N5/00—Machines for hulling, husking or cracking nuts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23N—MACHINES OR APPARATUS FOR TREATING HARVESTED FRUIT, VEGETABLES OR FLOWER BULBS IN BULK, NOT OTHERWISE PROVIDED FOR; PEELING VEGETABLES OR FRUIT IN BULK; APPARATUS FOR PREPARING ANIMAL FEEDING- STUFFS
- A23N5/00—Machines for hulling, husking or cracking nuts
- A23N5/08—Machines for hulling, husking or cracking nuts for removing fleshy or fibrous hulls of nuts
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
本发明涉及一株高产果胶酶的嗜气芽孢杆菌及其应用,该菌株于2023年09月13日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.28453。本发明所提供的嗜气芽孢杆菌不仅性状稳定遗传,具备较高的果胶酶活性,而且其具备优良耐受性能,可在高果胶果实加工中,应用于果实脱皮环节,可显著缩短脱皮周期,提升产品品质,而且对环境影响程度轻、无二次污染、工艺简单、方式灵活等优点,具有良好的开发应用前景。
Description
技术领域
本发明属于农产品加工技术领域,涉及一种芽孢杆菌及其应用,具体涉及一株高产果胶酶的嗜气芽孢杆菌及其在高果胶果实脱皮中的应用。
背景技术
果胶是一种天然的大分子多糖聚合物,由D-半乳糖醛酸通过α-1,4-糖苷键连接而成,其主要以果胶、原果胶、果胶酸的形式普遍存在于植物的果实、根、茎、叶中。果胶常与纤维素并存,构成植物细胞细胞壁及中间层粘结物,使得细胞间紧紧黏结,因此极大影响植物果实产品加工进程,降低处理效率,增大维护成本。
果胶酶(pectinase)属于一种复合酶,是一类能催化分解果胶质的酶的总称,广泛应用于果汁饮料、咖啡和胡椒脱皮等领域。尽管自然界中天然果胶酶广泛存在于动植物体内,但其产量低,不容易分离、提取与纯化,进而很难大规模生产。加之我国对果胶酶的研究起步较晚,酶生产成本高,难以形成大规模生产,不能满足现在食品工业需求,大部分果胶酶仍然需进口。
随着微生物酶技术的迅速发展,由于生长周期短,培养条件简单易控制,分布广等优势,已成为生产果胶酶的优良生物资源,主要包括芽孢杆菌、霉菌、酵母等。此类微生物能够产生相应的果胶酶来利用这类复杂的糖类物质,以维持自身的生长和繁殖。因此,研究相关的微生物裂解酶能拓展微生物代谢产物的应用,在提高果蔬产品加工效率具有重要意义。
高果胶果实,特别是胡椒,是重要的香辛料,是热带地区重要的经济作物,现今胡椒多以白胡椒产品的形式进入市场。然而,在胡椒加工过程中,胡椒果脱皮环节主要采用流水浸泡方式处理,虽然该工艺简单易行,但脱皮效率低,生产出的白胡椒异味重,品质低,并且存在废水排放量大,易污染环境等缺点。目前,如何高效快速实现高果胶果实脱皮、简化加工环节、生产优质农产品,是亟需解决的产业发展的现实问题之一。
发明内容
本发明旨在提供一株高产果胶酶的嗜气芽孢杆菌及其应用。
一株嗜气芽孢杆菌,于2023年09月13日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCC NO.28453。
本发明所提供的嗜气芽孢杆菌,筛选分离自新鲜胡椒果水浸泡去皮过程中,其表现出聚半乳糖醛酸、果胶裂解酶、果胶酯酶等酶活力,同时对高温、低酸环境以及胡椒精油与胡椒碱具有耐受性,且该性状稳定遗传。经16S rDNA序列鉴定,该菌株与已知芽孢杆菌相应序列的相似程度在99%以上,是嗜气芽孢杆菌的一个新菌株。在营养肉汤培养基上形态学特征为菌落颜色为乳白色,质地、形状扁平均一,不透明,表面及边缘整齐光滑,并且其显微镜观察细胞形态呈短杆状。
本发明的另一目的是提供所述嗜气芽孢杆菌在胡椒脱皮中的应用。
优选地,所述胡椒脱皮的方法,包括以下步骤:
以新鲜胡椒为原料,经挑选后,加入纯水,随后以106CFU/mL的量接种含所述嗜气芽孢杆菌的种子液进行水浸泡脱皮,过程中每天通气一次;浸泡结束后,清洗除杂得脱皮的产物,干燥得到产品。
优选地,所述纯水与果实的质量比例为:3:1~2(W/W)。
优选地,所述含嗜气芽孢杆菌的种子液的制备步骤如下:
将所述嗜气芽孢杆菌以1~5%的量接种至营养肉汤培养基中,于生化培养箱中35~38℃培养10~15h得到种子液。
优选地,所述的干燥是在60~70℃的条件下加热至产物含水量低于15%。
与现有技术相比,本发明的效果:
本发明所提供的嗜气芽孢杆菌筛选分离自新鲜胡椒果水浸泡去皮过程中,不仅性状稳定遗传,具备较高的果胶酶活性,而且其具备优良耐受性能,可在30~40℃环境下正常生长,在低pH(pH=5)条件下耐受性良好,可在高果胶果实加工中,应用于果实脱皮环节,可显著缩短脱皮周期,提升产品品质,而且对环境影响程度轻、无二次污染、工艺简单、方式灵活等优点,具有良好的开发应用前景。
附图说明
图1嗜气芽孢杆菌菌落特征图。
图2嗜气芽孢杆菌菌株形态图。
图3嗜气芽孢杆菌生长曲线图。
图4温度对嗜气芽孢杆菌生长及酶活的影响图。
图5pH对嗜气芽孢杆菌生长及酶活的影响图。
图6精油对嗜气芽孢杆菌生长及酶活的影响图。
图7胡椒碱对嗜气芽孢杆菌生长及酶活的影响图。
图8胡椒果水浸泡去皮过程中去皮率的变化。
图9胡椒果水浸泡去皮过程中酶活与pH的变化。
图10胡椒果自然水浸泡表皮显微结构变化。
图11胡椒果接种水浸泡表皮显微结构变化。
图12接种与自然浸泡白胡椒挥发性物质比较。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到,其中以分析纯试剂作为优选试剂。
实施例1菌株分离
取新鲜胡椒果进行水浸泡去皮,每天取浸泡水样品,进行梯度稀释,取3个适宜梯度涂布果胶琼脂培养基(5g/L果胶、10g/L蛋白胨、6g/L K2HPO4、2g/LKH2PO4、0.1g/LMgSO4、2g/L酵母提取物、20g/L琼脂)平板,然后将所有平板放入37℃恒温培养箱中倒置培养3~5d,长出肉眼可见的菌落后,将单菌落进行划线纯化,观察记录菌落的颜色及形态特征,同时经镜检为纯种后利用甘油冷冻保藏法进行保藏。将灭菌后的50%甘油与纯培养得到的菌液进行1:1(V/V)混合,于-80℃冰箱中冻藏保存备用。
总共分离出50株不同类型的菌株,进一步将菌株进行筛选,获得具有果胶酶活性的菌株。
实施例2菌株的筛选
(1)初筛:将分离菌株活化后,点接于果胶琼脂培养基的平板上,于28℃培养3~5d。用游标卡尺测量单菌落的直径(d),利用刚果红染色法对果胶平板进行染色,观察透明圈的大小并记录,透明圈直径为D,菌落直径为d,得到D/d的值。染色方法是将刚果红染液倾倒在有单菌落的果胶平板上没过平板空白处,染色20min后,倒掉,倾倒1mol/L的NaCl洗液于平板上20min后倒去洗液,重复洗一次。
所述刚果红染液为:称取刚果红染色试剂7.000g,溶解,蒸馏水定容至1L,配制1mol/L染液。
(2)复筛:将初筛出的菌株进行复筛,菌株活化后以1%的接种量接种至100mL的营养肉汤中,于37℃培养3d,采用DNS法测菌株产果胶酶的活力。取1mL发酵液在10000r/min,4℃条件下离心5min,取上清液稀释适当倍数作为粗酶液。为酶活组、灭活组2组,每组试管编号A与B。每个试管分别加入2.5mL 1.0%果胶溶液于50℃水浴预热5min,A管中加入0.5mL粗酶液,B管加入0.5mL的灭活粗酶液(沸水浴5min),试管均放入50℃水浴反应30min。取1.0mL反应液于试管中,再加入1.0mL DNS试剂,沸水浴10min。取出后用迅速冷却至室温,加蒸馏水稀释适宜倍数,摇匀,于在540nm处测定OD值,酶活力计算公式:
A:酶样吸光度;A0:酶空白样吸光度;N:酶液稀释倍数;3:测定酶活时,取了反应液的1/3;1000:1mgD-半乳糖醛酸为1000μg;K:标准曲线的斜率;t:反应时间。
所述1.0%果胶溶液为:用pH为5的柠檬酸-磷酸氢二钠的缓冲液配制1g/L的果胶底物,4℃保存,有效期3天。
所述DNS试剂为:取3,5-二硝基水杨酸10g,加入2mol/L氢氧化钠溶液200mL,将3,5-二硝基水杨酸溶解,然后加入酒石酸钾钠300g,待其完全溶解,用去离子水稀释至2000mL,棕色瓶保存。
刚果红作为一种染料,能与大分子多聚糖结合成红色复合物,而不与二糖和单糖结合,当分泌在菌落周围培养基的果胶酶将多聚糖果胶降解为小分子糖后,NaCl洗液就能够使结合不牢固的果胶洗去,菌落周围就能够出现透明圈,D/d值越大酶活越高。通过D/d值的大小筛选出10株菌株进行下一步复筛。
以果胶为底物,以发酵液为粗酶液,在一定温度下反应30min。以半乳糖醛酸作为反应产物,采用DNS法测定半乳糖醛酸含量,由此计算酶活。酶活单位定义为:在50℃,pH5.0条件下,每分钟水解果胶产生1μg半乳糖醛酸定义为一个酶活单位(U)。结果见表1,结果显示仅有部分芽孢杆菌具有一定的果胶酶活性,并且不同菌株的果胶酶活性差异显著,尤其是菌株P01酶活力高达2.73U/mL,而菌株J08、J20、P09、P25也具备一定酶活性,但相对较低,因此选择菌株P01进行先一步研究。
表1菌株酶活性
注:“-”表示未检出酶活性。
菌株P01在营养肉汤培养基上的菌落形态如图1和2所示。由图中可以看出,菌株P01在营养肉汤培养基上形态学特征为菌落颜色为乳白色,质地、形状扁平均一,不透明,表面及边缘整齐光滑;并且其显微镜观察细胞形态呈短杆状。
实施例3菌株的鉴定
采用Ezup柱式细菌基因组DNA抽提试剂盒对菌株P01 DNA进行提取(参照产品说明书),于-20℃保存备用。使用凝胶回收试剂盒回收PCR产物目的片段,送生工生物工程(上海)股份有限公司进行基因测序。
利用BLAST软件在GenBank核苷酸序列数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)中进行同源序列搜索,比较供试菌株(菌株P01)与已知芽孢杆菌相应序列的相似程度,通过与已知芽孢杆菌相应序列的比较,对供试菌株的芽孢杆菌的种属进行鉴定(相似程度在99%以上的视为同一个种)。鉴定结果显示,供试菌株(菌株P01)与已知芽孢杆菌相应序列的相似程度在99%以上,是嗜气芽孢杆菌的一个新菌株。
菌株P01,保藏编号为CGMCC NO.28453,其16S rDNA基因序列如SEQ ID NO.1所示。
SEQ ID NO.1序列:TGGCCAAGGCTAGCTGACGACGTAGATTAGAGTCTGAGCGTGA CGAGAAGGAGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAGTAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTCTATGGTAACAGCGACTAGCAGGAGTTCTGCTC。
实施例4菌株P01生长曲线
菌株P01生长曲线:将菌株P01以106CFU/mL的接种量接种于200mL营养肉汤培养基中,37℃静置培养,每隔一定时间测定一次培养液的OD600nm,绘制芽孢杆菌生长曲线,如图3所示。随着时间的推移,菌株P01具有最强的生长能力,菌株P01延滞期较短,小于2h;2~12h基本进入对数生长期,此时菌株迅速生长,菌株生长速率显著高于死亡速率,并且此时菌株活力高适合用于接种液;12h后进入稳定期且在36h后个别温度下菌株已经开始进入衰退期。因此,确定菌株适宜接种时间为10h以及“收获”时间为24h。
实施例5菌株P01生长特性评价
将菌株P01进行耐受性研究,将活化的种子液以106CFU/mL的接种量分别接种于50mL不同处理(包括不同温度、pH以及不同浓度的胡椒精油和胡椒碱)的营养肉汤培养基,于37℃条件下静置培养48h,测定其在600nm处的OD值及发酵液果胶酶活性,实验设置三个重复,进一步评价该菌株的温度受性、胡椒精油耐受性、胡椒碱耐受性、pH耐受性等性能。
虽然菌株P01分离自胡椒浸泡过程中,但其不可避免地会受到多种环境因素的胁迫,最常见的如温度和pH值,以及胡椒中自身的精油与胡椒碱。这些环境胁迫可能通过抑制菌株的生长代谢,导致脱皮时间延长甚至停滞,从而影响着白胡椒的质量。因此对菌株P01进行温度、pH以及精油与胡椒碱的耐受性研究,结果见图4-7。
从图中可以看出,相比其它温度,菌株P01在50℃的高温环境下生长较为缓慢,此时果胶酶活力也相对较低,然而随着温度的降低其生长速率和酶活逐渐升高,尤其在30℃时达到最大;pH对菌株P01的生长与酶活影响显著,在pH=5时菌株生长状态也相对较好;当精油浓度为0.05%时P01的长势也相对较好,但随着精油用量的继续增加,菌株P01的生长被抑制,胡椒的精油含量在0.01~0.01%之间,菌株P01仍然能够适应此环境;菌株P01对浓度高于0.05%的胡椒碱敏感。
综上,本发明所提供的菌株P01能够较好适应胡椒浸泡过程中的多种逆境胁迫问题,应用前景好。
实施例6菌株P01在胡椒果去皮中的效果
试验组(接种浸泡):以新鲜胡椒果为原料,经挑选后,加入3倍质量的纯水,随后以106CFU/mL的量接种菌株P01的种子液(将菌株P01以1%的量接种至100mL营养肉汤培养基中,于生化培养箱中37℃培养12h得到种子液)进行水浸泡去皮,过程中每天通气一次;浸泡结束后,清洗除杂得脱皮的胡椒粒,60~70℃下干燥使胡椒粒的含水量低于12~15%,得到白胡椒。
常规处理组(自然浸泡):以新鲜胡椒果为原料,经挑选后,加入3倍质量的纯水进行水浸泡去皮,过程中每天通气一次;浸泡结束后,清洗除杂得脱皮的胡椒粒,60~70℃下干燥使胡椒粒的含水量低于12~15%,得到白胡椒。
测定脱皮率、pH、酶活,白胡椒白度、硬度、精油、胡椒碱、挥发性化合物以及感官评价。
接种菌株P01的胡椒去皮程中理化指标结果见表2,试验组与常规处理组相比,白胡椒表现出明显优势。
表2不同处理胡椒去皮过程中基本理化结果
参见图8-11,由图8-9所示,随着脱皮过程的进行,相比常规处理组(自然浸泡),接种菌株P01后(接种浸泡)胡椒果的脱皮速率显著加快,其在4d后脱皮率已经达到80%以上,表明接种菌株P01后可显著缩短胡椒的脱皮周期。并且随着浸泡的进行表皮发生显著裱花,图10-(a)与11-(a)新鲜胡椒果的表皮显微结构图,由图中可以清楚的看到胡椒果肉的细胞结构及一些亚显微结构,细胞结构比较完整;而随着水浸泡的进行,在第4天后接种菌株P01的胡椒的果肉结构变得不平整,植物细胞的一些基本结构遭到破坏,细胞内分区结构被打破,果肉组织全部崩解浆化(图11-(b))。试验组(接种浸泡)和常规处理组(自然浸泡)的pH值均呈下降趋势,而接种菌株P01后其pH快速下降,在3d后保持稳定(pH为5.5),而果胶酶的pH作用范围为2.5~6之间,因此该pH环境下能更好适应果胶酶发挥作用。同时,对比两种处理方式,其白胡椒的精油和胡椒碱含量并无显著差异,而胡椒白度值显著得到提升。因此可以看出本发明所提供的菌株P01在白胡椒生产加工中具有较大应用前景。
感官品评由10位感官评定员组成,在实验前,经过培训后的评定员根据参考标准进行打分,结果取10人分数的均值。感官评价参考标准为:胡椒辛辣味突出且无异味6~9分、胡椒辛辣平淡淡且轻微异味4~5分、胡椒辛辣味不足且异味浓郁1~3分。不同处理方式白胡椒感官评价结果见表3,其不同处理方式感官得分差异显著,相比常规处理组,试验组得分较高,表明其异味明显降低,常规处理组得分均较低,表明异味较重。研究表明白胡椒异味主要可能是由于3-甲基吲哚、甲基苯酚等粪臭素物质所致。
表3不同处理白胡椒异味物质及气味强度
注:”-“表示未检出
采用离子迁移色谱(GC-IMS)对比了两种加工方式白胡椒的挥发性物质的差异,如图12所示,接种浸泡与自然浸泡所得白胡椒挥发性物质差异显著,共初步定性出51种挥发性化合物。相比于自然浸泡,接种浸泡虽然部分物质相对丰度较低,但是2-茨醇、β-蒎烯、β-罗勒烯、异松油烯、α-松油醇、苯乙醛等胡椒中特征香气物质丰度相对较高。而自然浸泡所得的白胡椒中突出了丁酸、异戊酸、甲基苯酚等物质,有研究表明这类物质是给胡椒带来“粪臭味、丁酸臭”的主要物质。进一步通过GC-MS测定发现,主要异味物质为3-甲基吲哚、4-甲基苯酚、苯酚、3-甲基苯酚、异丁酸、丁酸、戊酸等异味物质,其中3-甲基吲哚与4-甲基苯酚是白胡椒中体现为粪便臭的主要挥发性物质,相比自然浸泡组其含量显著减低;此外,接种浸泡的白胡椒中并未检出能够产生汗臭的戊酸与异丁酸。由此可见,进一步从物质的角度说明菌株P01能够有效降低胡椒异味的产生,并且可加快去皮过程。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一株嗜气芽孢杆菌,保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏编号为CGMCCNO.28453。
2.权利要求1所述的嗜气芽孢杆菌在胡椒脱皮中的应用。
3.根据权利要求2所述的应用,其特征在于,所述胡椒脱皮的方法,包括以下步骤:
以新鲜胡椒为原料,经挑选后,加入纯水,随后以106CFU/mL的量接种含所述嗜气芽孢杆菌的种子液进行水浸泡脱皮,过程中每天通气一次;浸泡结束后,清洗除杂得脱皮的产物,干燥得到产品。
4.根据权利要求3所述的应用,其特征在于,所述纯水与果实的质量比为:3:1~2。
5.根据权利要求3所述的应用,其特征在于,所述嗜气芽孢杆菌的种子液的制备步骤如下:
将所述嗜气芽孢杆菌以1~5%的量接种至营养肉汤培养基中,于生化培养箱中35~38℃培养10~15h得到种子液。
6.根据权利要求3所述的应用,其特征在于,所述的干燥是在60~70℃的条件下加热至产物含水量低于15%。
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