CN115305217B - 一株降解亚硝酸盐、抗氧化和产香的植物乳杆菌及应用 - Google Patents
一株降解亚硝酸盐、抗氧化和产香的植物乳杆菌及应用 Download PDFInfo
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- CN115305217B CN115305217B CN202210765438.3A CN202210765438A CN115305217B CN 115305217 B CN115305217 B CN 115305217B CN 202210765438 A CN202210765438 A CN 202210765438A CN 115305217 B CN115305217 B CN 115305217B
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- lactobacillus plantarum
- nitrite
- sausage
- strain
- degrading
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Classifications
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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
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- A—HUMAN NECESSITIES
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- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
- A23L13/45—Addition of, or treatment with, microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/60—Comminuted or emulsified meat products, e.g. sausages; Reformed meat from comminuted meat product
- A23L13/65—Sausages
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- A—HUMAN NECESSITIES
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- A23V2400/00—Lactic or propionic acid bacteria
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Abstract
本发明公开一株降解亚硝酸盐、抗氧化和产香的植物乳杆菌及应用，属于微生物领域，所述菌株为植物乳杆菌Lactobacillusplantarum WCFS1，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:M2021228。本发明公开的菌株可以用于降解亚硝酸盐、并降低亚硝酸盐含量；同时，该菌株具有良好的抗氧化和产香特性，对发酵肉制品风味形成、贮藏抗氧化能力及抑制亚硝酸盐残留，确保中式发酵肉制品安全及品质，提供良好保障。该菌株同时对酸、牛胆盐、亚硝酸盐及NaCl均具有较好的耐受性，适应发酵肉制品生产条件，并能够保持良好活性，具有肉制品发酵菌株必备条件。
Description
技术领域
本发明属于微生物新品种技术领域，具体涉及一株降解亚硝酸盐、抗氧化和产香的植物乳杆菌及应用。
背景技术
发酵肉制品风味独特、保存期长、营养丰富，深受消费者喜爱。但在发酵肉制品加工贮藏过程中容易受外界条件影响，发生过度氧化，亚硝酸盐积累导致产品品质劣化、风味改变。因此，提供一株具有降解亚硝酸盐、抗氧化和产香能力的功能性肉用植物乳杆菌，确保中式发酵肉制品安全及品质，提供良好保障,在肉用食品中具有一定的可行性和现实意义。
亚硝酸盐多用于传统腌肉制品中，起到减少水分、降低水份活度和保鲜的作用，并且具有发色、抑菌、增味和抗氧化等重要作用，常作为固化剂和防腐剂。据世界卫生组织报道，由饮食平均每天摄入的硝酸盐含量通常在43～141mg。亚硝酸盐使用量虽然循国标进行添加，但是仍会出现残留量超标问题，并且亚硝酸盐是一种潜在的致癌物质，在体内的残留和积累都会造成一定的伤害，具有潜在的食品安全风险，这导致其应用受到很大限制。因此，亚硝酸盐在食品中的残留问题一直是受大众关注。目前针对减少肉制品中亚硝酸盐含量的方法，主要包括：物理降解法、化学降解法和生物降解法。物理化学降解法主要是对原料进行前处理，如：Maristela研究添加胡萝卜和甜菜根粉作为亚硝酸盐替代品在发酵干香肠中的应用，结果发现添加甜菜根粉的添加影响发酵干香肠的色泽，相比较萝卜粉的添加是作为亚硝酸盐替代品的最佳选择(Maristela Midori Ozaki,Paulo E.S.Munekata,Rickyn Alexander Jacinto-Valderrama,et al.Beetroot and radish powders asnatural nitrite source for fermented dry sausages.2021,171.)。Branislav研究采用超临界流体萃取番茄渣和有机薄荷精油，将番茄渣和有机薄荷精油添加到煮熟的猪肉香肠中，发现番茄渣和有机薄荷精油可作为亚硝酸钠在食品中的部分替代物，添加到煮熟的猪肉香肠中，可减少亚硝酸钠的残留(YanHua Tian,HuiZhi Du,LiWang,et al.NitriteScavenging and Inhibition of N-Nitrosamines Formation by Phenolic ExtractsFrom Diospyros lotus L.Leaves and Active Ingredients.2020,15(9).)。其中生物降解法主要为添加亚硝酸盐降解发酵剂，具有高效健康，提升肉制品风味的优势，是目前的研究热点。而生物法中最常用的发酵剂是乳酸菌，乳酸菌在发酵过程中其代谢产物如乳酸可改变发酵环境、亚硝酸盐还原酶可有效降解亚硝酸盐(Tocheva,Elitza I,Resell,etal.Side-On Copper-Nitrosyl Coordination by Nitrite Reductase.[J].Science,2004.)，达到高效降解亚硝酸盐的效果。
肉制品中含有丰富的脂肪和蛋白质，在光、热、酶等条件下肉制品易发生氧化反应，是在加工肉制品生产中的关键问题。脂肪氧化反应不断进行，直到不饱和脂肪酸和氧气被耗气才会终止，同时脂肪氧化产生的自由基会引发蛋白质氧化，导致肉制品营养价值降低，破坏风味。目前，在肉制品加工过程中加入抗氧化剂以延缓氧化，使用天然抗氧化剂(Cheng J.,Xu L.,Xiang R.,et al.Effects of mulberry polyphenols on oxidationstability of sarcoplasmic and myofibrillar proteins in dried minced porkslices during processing and storage[J].Meat science,2020,160:107973.李文慧,刘飞,李应彪,等.植物多酚对肉制品蛋白氧化的抑制机理及其延长货架期的应用[J].食品科学,2019,40(21):266-272.SadeghinejadN.,Amini Sarteshnizi R.,Ahmadi GavlighiH.,et al.Pistachio green hull extract as a natural antioxidant in beefpatties:Effect on lipid and protein oxidation,color deterioration,andmicrobial stability during chilled storage[J].LWT,2019,102:393-402.陈洪生,牛百慧,刘欢,等.4种香辛料提取物对猪肉肌原纤维蛋白抗氧化特性的影响[J].食品科学,2019,40(4):95-101)、微生物抗氧化剂(Pagels F.,Guedes A.C.,Amaro H.M.etal.Phycobiliproteins from cyanobacteria:Chemistry and biotechnologicalapplications[J].Biotechnology Advances,2019,37(3):422-443.王秀丽,陆晨红,霍俊辉,等.接种发酵对发酵牛肉香肠品质及多肽抗氧化活性的影响[J].肉类研究,2022,36(01):7-13.蔡永敏,黄海英,胡炜东,等.混合发酵剂的筛选及对发酵猪肉香肠加工特性及安全性能的影响[J].食品科技,2022,47(01):138-144.)。
[1]其中，将具有抗氧化能力的乳酸菌(Montanari,Chiara,Gatto,et al.Effectsof the diameter on physico-chemical,microbiological and volatile profile indry fermented sausages produced with two different starter cultures[J].FoodBioscience on Sciverse Sciencedirect,2018(22):9-18.Paik H D,Lee JY.Investigation of reduction and tolerance capability of lactic acid bacteriaisolated from kimchi against nitrate and nitrite in fermented sausagecondition[J].Meat Science,2014,97(4):609-614.Bartkiene E,Bartkevics V,Mozuriene E,et al.The impact of lactic acid bacteria with antimicrobialproperties on biodegradation of polycyclic aromatic hydrocarbons and biogenicamines in cold smoked pork sausages[J].Food Control,2017(71):285-292.Chen Q,Kong B,Han Q,et al.The role of bacterial fermentation in the hydrolysis andoxidation of sarcoplasmic and myofibrillar proteins in Harbin dry sausages[J].Meat Science,2016,121(11):196-206.)应用到肉制品加工中，具有提升营养价值、促进产品风味形成、延缓蛋白氧化等优点。
发明内容
针对上述发酵中式香肠过程中，亚硝酸盐含量高，蛋白质和脂肪氧化过度，不仅降低香肠的营养价值，而且产生了对人体有害物质，对此，发明人通过对香肠中的乳酸菌诱变，并筛选出突变菌株的方法，得到了一株降解亚硝酸盐、抗氧化和产香的植物乳杆菌，所述植物乳杆菌的名称为植物乳杆菌Lactobacillus plantarum WCFS1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2021228。
其中，所述植物乳杆菌Lactobacillus plantarum WCFS1的培养基为MRS肉汤培养基。
其中，所述MRS肉汤培养基成分包括：蛋白胨、牛肉粉、酵母粉、葡萄糖、吐温80、磷酸氢二钾、乙酸钠、柠檬酸氢二铵、硫酸镁、硫酸锰。
所述植物乳杆菌Lactobacillus plantarum WCFS1对酸、牛胆盐、亚硝酸盐及NaCl的环境具有耐受性，在此环境中可以降解亚硝酸盐，降低环境中亚硝酸盐的含量，同时具有抗氧化作用，延缓脂肪和蛋白的氧化。
所述植物乳杆菌Lactobacillus plantarum WCFS1用于制备香肠，降低香肠腌制过程中亚硝酸盐含量，同时延缓香肠脂肪和蛋白质氧化，并提高香气物质的产生量。
有益效果
本发明保藏的植物乳杆菌Lactobacillus plantarum WCFS1同时具有抗氧化性、降低环境中亚硝酸盐和耐受其中，本发明保藏的植物乳杆菌Lactobacillus plantarumWCFS1抗氧化能力体现在：T-AOC为30.85±0.67U/mL，羟自由基能力力清除率可达到96.23±1.22％；超氧阴离子清除率可达到91.95±0.53％；DPPH清除率可达到60.81±0.46％。将该菌株接种到中式发酵香肠中发现，该菌株的添加能够有效延缓香肠脂肪和蛋白质氧化。能够显著减缓香肠TBARs值，发酵前期显著降低POV值和LOX酶活性(p<0.05)，说明该菌株能够延缓香肠中脂肪氧化；在发酵过程中该菌株还能够抑制香肠肌浆蛋白与肌原纤维蛋白的羰基形成，减缓了其巯基的流失，同时降低了蛋白质二级结构α-螺旋结构相对含量的损失，减少蛋白表面疏水基团的暴露，表明菌株具有良好的延缓香肠中蛋白质氧化的效果。
本发明保藏的植物乳杆菌Lactobacillus plantarum WCFS1在低浓度NaNO2下FQR降解率可达94.85±2.96％，亚硝酸盐还原酶活力为174.60±13.67U/104cell。可以有效降低香肠中亚硝酸盐的含量。在酸、牛胆盐、亚硝酸盐及NaCl外界环境刺激下，菌株仍具有降解亚硝酸盐和抗氧化的能力。这些是之前的乳酸菌均没有报道的，改环境刚好适用于腌制中式香肠的环境，在酸、牛胆盐、亚硝酸盐及NaCl的环境中，降低亚硝酸盐的同时，延缓香肠蛋白质和脂肪的氧化，增强香肠的保质期，同时增加香肠的风味。之前没有同时具备此特性的乳酸菌。
本发明保藏的植物乳杆菌Lactobacillus plantarum WCFS1在发酵过程中形成特殊的风味，GC-MS气质联用共检测到153种挥发性物质，香肠中酯类物质种类由16种增加到20种，酯类的总相对含量增加了17.13％，尤其是己酸乙酯(增加了15.32％)、癸酸乙酯(增加了39.82％)、壬酸乙酯(增加了52.63％)、辛酸乙酯(增加了50％)和(E)-2-甲基-2-丁烯酸己酯(增加了73.62％)等特征风味物质，说明该菌株有助于传统中式香肠风味物质的产生。
本发明保藏的乳酸菌抗恶劣环境的能力强，对发酵肉制品风味形成、贮藏抗氧化能力及抑制亚硝酸盐残留，确保中式发酵肉制品安全及品质，提供良好保障。该菌株同时对酸、牛胆盐、亚硝酸盐及NaCl均具有较好的耐受性，适应发酵肉制品生产条件，并能够保持良好活性，具有肉制品发酵菌株必备条件。
附图说明
图1植物乳杆菌FQR菌株菌落示意图
图2植物乳杆菌FQR菌株形态示意图
图3不同浓度NaNO2对植物乳杆菌FQR降解能力的影响
图4不同浓度NaNO2对植物乳杆菌FQR亚硝酸盐还原酶活性的影响
图5不同pH值下植物乳杆菌FQR对亚硝酸盐降解能力和亚硝酸盐还原酶活性
图6不同胆盐含量下植物乳杆菌FQR对亚硝酸盐降解能力和亚硝酸盐还原酶活
图7不同NaNO2含量下植物乳杆菌FQR对亚硝酸盐降解能力合亚硝酸盐还原酶活性
图8不同NaCl含量下植物乳杆菌FQR对亚硝酸盐降解能力和亚硝酸盐还原酶活性
图9发酵香肠中挥发性物质相对含量的变化
具体实施方式
下面结合实施例，对本发明提出的保藏的植物乳杆菌Lactobacillus plantarumWCFS1及其特性作进一步地描述。
实施例1
植物乳杆菌Lactobacillus plantarum WCFS1的鉴定及保藏。
发明人对传统发酵香肠中乳酸菌进行诱变处理，从中选取突变菌株分离纯化得到的纯种的菌株，并对其进行革兰氏染色实验、形态鉴定和基因序列进行鉴定，具体方法如下和结果如下：
(1)革兰氏染色实验
1)涂片；2)固定；3)初染：结晶紫覆盖染色1min；4)水洗：至无色为止；5)媒染：碘液覆盖染色1min；6)水洗；7)脱色：倾斜玻片，滴加95％乙醇，冲洗30s；8)水洗；9)复染：滴加沙黄覆盖染色1-2min；10)水洗；11)干燥镜检观察。革兰氏阳性菌菌体呈现紫色，革兰氏阴性菌呈现红色。
(2)菌种形态
革兰氏染色试验后在油镜下观察，记录实验结果。
(3)基因序列
按照TaKaRa MiniBEST Bacteria Genomic 3.0试剂盒操作说明书，提取植物乳杆菌FQR基因组总DNA，进行分子生物学鉴定：以菌株DNA为模板进行PCR扩增。PCR扩增上游引物其核苷酸序列5-TGATCCTGGCTCAGGACGAA-3，下游引物其核苷酸序列5-TGCAAGCACCAATCAATACCA-3，送于北京华大基因研究中心16S rRNA测序，要求测通。将拼接后的基因序列与GenBank数据库进行序列比对分析，进行同源性比较。
检测结果与分析
形态学特征
在37℃，MRS肉汤培养基中培养48h后，植物乳杆菌FQR菌落呈现圆形、隆起、乳白色、表面光滑，如图1所示。所述的植物乳杆菌Lactobacillus plantarum WCFS1的MRS肉汤培养基成分(g/L)：10g蛋白胨、8g牛肉粉、4g酵母粉、20g葡萄糖、1.0mL吐温80、2g磷酸氢二钾、5g乙酸钠、2g柠檬酸氢二铵、0.2g硫酸镁、0.04g硫酸锰，加蒸馏水至1000mL。
所述的植物乳杆菌Lactobacillus plantarum WCFS1的MRS固体培养基成分，是在植物乳杆菌Lactobacillus plantarum WCFS1的MRS肉汤培养基的基础上添加15g琼脂制成。
用革兰氏染色进行染色，镜检结果：革兰氏染色呈紫色，是革兰氏阳性菌。菌株形态为直或弯曲杆状，为单个、成对或成链，如图2所示。
乳酸菌菌株为植物乳杆菌Lactobacillus plantarum WCFS1，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:M2021228(植物乳杆菌FQR)，保藏地址为中国.武汉.武汉大学.保藏日期为2021年3月15日。
实施例2
植物乳杆菌Lactobacillus plantarum WCFS1FQR的生理生化特性试验
(a)产粘性试验
以1‰的接种量将FQR接种于产黏性培养基中，37℃培养24h后，用接种针挑取生长较好的单个菌落进行观察，观察该菌是否具有产粘性。
(b)产H2O2试验
以1‰的接种量将FQR接种于产H2O2培养基中，37℃连续培养5d，观察培养基中各个菌落附近MnO2黑色是否溶解，若溶解则为阳性，说明该菌株产H2O2。
(c)产氨试验
以1‰的接种量将FQR接种于产氨培养基中，37℃培养24h后，滴入3至5滴氨试剂，与未接种产氨培养基进行对照，若产生了红棕色沉淀或者黄色沉淀，表明其为阳性，反之则是阴性。
(d)葡萄糖产气试验
以1‰的接种量将FQR接种于葡萄糖产气培养基内(此操作于杜氏小管中进行)，37℃下培养24h，未接种的培养基为空白对照组，观察有无气泡生成。
(e)产H2S试验
以1‰的接种量将FQR接种于FeCl2培养基培养基内，37℃下连续培养24h后观察试验结果，若是试管内有黑色的沉淀线出现，则为阳性，表明产生了H2S。
(f)氨基酸脱羧酶试验
以1‰的接种量将FQR接种于氨基酸脱羧酶培养基中，37℃培养24h，以未添加氨基酸的MRS液体培养基作为空白对照，观察现象，若培养基颜色为紫色表明其为氨基酸脱羧酶试验阳性；若培养基颜色为黄色则表明为阴性。
(g)耐盐性试验
培养基的配制：在MRS液体培养基的基础上添加4％、6％NaCl。以1‰的接种量将FQR分别接种于含4％、6％NaCl的MRS液体培养基中，37℃培养24h，观察培养基是否浑浊。
(h)耐亚硝酸盐试验
培养基的配制：在MRS培养基的基础上添加150mg/kgNaNO2。
以1‰的接种量将FQR接种于含150mg/kg NaNO2的MRS液体培养基中，37℃培养24h，观察培养基是否浑浊。
检测结果与分析
植物乳杆菌FQR具有乳酸菌的基本特性：耐150mg/kgNaNO2，不产粘性，不具有氨基酸脱羧酶活性，不产氨，耐4％NaCl，发酵葡萄糖不产气，不产H2O2，不产H2S。
实施例3
植物乳杆菌Lactobacillus plantarum WCFS1亚硝酸盐降解能力的测定方法及结果
(1)亚硝酸盐含量的测定方法：培养基置于离心管中3500×g离心5min，取上清，不经过提取液净化的步骤，直接加2mL对氨基苯磺酸溶液，混匀，静置3～5min后各加入1mL盐酸萘乙二胺溶液(2g/L)，静置15min，于波长538nm处测定吸光度值。同时做试剂空白。NaNO2含量计算公式如下：
式中：X—试样中NaNO2的含量，mg/kg；A1—测定用样液中NaNO2的质量，μg；m—试样质量，g；V1—测定用样液体积，mL；V0试样处理液总体积，mL。
(2)亚硝酸盐降解率测定：以2％接种量接入含有0～0.225％NaNO2 MRS肉汤培养基中，37℃培养16h。测定培养基中NaNO2含量，并取菌液利用稀释涂布平板法记录活菌数，每组三个重复。NaNO2降解率(degradation rate，DR)公式如下：DR(％)＝(1-X2/X1)×100
式中：X1—原始培养基中NaNO2的含量，单位为毫克每千克(mg/kg)；X2—培养16h后培养基中NaNO2的含量，单位为毫克每千克(mg/kg)。
检测结果分析
植物乳杆菌FQR降解NaNO2的能力，如图3-a所示，在0.045～0.225％NaNO2范围内，随着MRS肉汤培养基中NaNO2含量的增加，FQR降解NaNO2能力呈下降趋势。FQR于0.045％和0.075％NaNO2 MRS中降解率无显著差异，分别为94.85±2.96％和94.18±3.87％，而在0.225％NaNO2浓度时，降解率仅为7.05±2.77％，同时，取100uL菌液涂布于MRS固体培养基上，结果发现无活菌存在(图3-b)。说明在0～0.075％NaNO2范围内，FQR具有良好的降解NaNO2能力，而高浓度的NaNO2对植物乳杆菌FQR降解NaNO2能力具有抑制作用。这是由于NaNO2浓度过高抑制植物乳杆菌FQR的活性，导致降解NaNO2能力下降。
实施例4
植物乳杆菌Lactobacillus plantarum WCFS1亚硝酸盐还原酶活性的测定方法及结果
以2％接种量接入含有0～0.150％NaNO2 MRS肉汤培养基中，37℃培养16h。培养液于4℃条件下3500×g离心10min，弃上清，收集菌体，用0.02M磷酸盐缓冲液(PBS，pH＝7.2)洗涤两次后重悬于PBS，置于冰上超声波破碎细胞，超声波细胞破碎仪工作参数为：功率300W，超声3s，间隔7s，总时长3min。破碎细胞液于4℃条件下10000×g离心10min，取上清置于冰上，利用亚硝酸还原酶试剂盒测定酶活。酶活单位定义为：每104个细胞每小时还原1μmol NO2 -的量为一个酶活单位。
检测结果分析
不同浓度NaNO2对FQR亚硝酸盐还原酶活性的影响，如图4所示，在0～0.150％NaNO2范围内，NiR活性呈下降趋势，但0.075％NaNO2时NiR活力为119.68±9.03U/104cell，相对于0.045％NaNO2条件酶活力下降了31.45％。FQR于空白和0.045％NaNO2 MRS中NiR活力差异不显著，分别为181.55±8.63U/104cell和174.60±13.67U/104cell，说明0～0.045％NaNO2浓度范围内，NiR活力没有受到显著影响。
实施例5
植物乳杆菌Lactobacillus plantarum WCFS1抗氧化能力的测定方法及结果
以1％接种量将活化好的菌株接种至MRS肉汤中，置于37℃培养18h，于4℃，4500rpm/min条件下离心10min，收集上清液即为发酵液。测定发酵液的总抗氧化能力、羟自由基清除能力、超氧阴离子清除能力和DPPH自由基清除能力。
检测结果分析
植物乳杆菌FQR发酵液具有良好的抗氧化能力，T-AOC为30.85±0.67U/mL，羟自由基能力力清除率可达到96.23±1.22％，超氧阴离子清除率可达到91.95±0.53％，DPPH清除率可达到60.81±0.46％。
实施例6
植物乳杆菌Lactobacillus plantarum WCFS1对中式发酵香肠中脂肪氧化和蛋白氧化的测定方法及结果
中式香肠的制作工艺为：将70％的猪瘦肉和30％的猪肥肉切碎，按照比例添加以下食材：盐(3％，w/w)，糖(7％，w/w)，谷氨酸钠(0.2％，w/w)，五香粉(0.1％，w/w)，姜粉(0.15％，w/w)，大曲酒(2％，v/w)，红曲粉(0.25％，w/w)，水(10％，w/w)，接种量为为5.0×105～3.0×106CFU/g。混合均匀后灌肠，在15℃培养箱中发酵。
(1)参照GB 5009.181-2016食品安全国家标准食品中丙二醛的测定、GB5009.227-2016食品安全国家标准食品中过氧化值的测定和脂肪氧合酶活性测定试剂盒操作，测定添加植物乳杆菌对脂肪氧化的影响。
(2)参照Yu(Yu D.,Feng M.Q.,Sun J.,et al.Protein degradation andpeptide formation with antioxidant activity in pork protein extractsinoculated with Lactobacillus plantarum and Staphylococcus simulans[J].Meatscience,2020,160:107958.)提取香肠中的蛋白质，测定其羰基含量、巯基含量和傅里叶红外光谱，分析添加植物乳杆菌对蛋白氧化的影响。
检测结果分析
添加植物乳杆菌具有显著降低TBARs值的作用(p<0.05)，在发酵前期可显著降低POV值和LOX酶活性(p<0.05)，说明该菌株能够延缓香肠中脂肪氧化。在发酵过程中该菌株还能够抑制香肠肌浆蛋白与肌原纤维蛋白的羰基形成，减缓了巯基的流失，同时降低了蛋白质二级结构α-螺旋结构相对含量的损失，减少蛋白表面疏水基团的暴露，表明菌株具有良好的延缓香肠中蛋白质氧化的效果。
实施例7
植物乳杆菌Lactobacillus plantarum WCFS1对中式发酵香肠中挥发性物质的测定方法及结果。
中式香肠的制作工艺为：将70％的猪瘦肉和30％的猪肥肉切碎，按照比例添加以下食材：盐(3％，w/w)，糖(7％，w/w)，谷氨酸钠(0.2％，w/w)，五香粉(0.1％，w/w)，姜粉(0.15％，w/w)，大曲酒(2％，v/w)，红曲粉(0.25％，w/w)，水(10％，w/w)，接种量为为5.0×105～3.0×106CFU/g。混合均匀后灌肠。
参照曹辰辰(曹辰辰,冯美琴,孙健,徐幸莲,周光宏.功能性发酵剂对发酵香肠氧化稳定性及挥发性风味物质的影响[J].食品科学,2019,40(20):106-113.)等以固相微萃取法处理香肠样品。取香肠切碎，称取5g装入20mL顶空瓶中压紧至瓶身1/3处，加入环己酮为内标物，将老化后的萃取头插入样品瓶的顶空部分，80℃吸附60min，将吸附后的萃取头取出插入气相色谱进样口，于250℃解析10min，采集数据，50min后完成采集。色谱条件：使用TR-5MS毛细管柱(30m×0.25mm×0.25μm)，以氦气为载气，流速1mL/min。升温程序：炉温在40℃下保持3min，以5℃/min的升温速率升至90℃，不保持，再以10℃/min的速率升温至230℃，保持6min。质谱条件：离子源温度200℃，电离方式EI+，电子能量70eV，发射电流120μA，扫描质量范围30～50m/z。根据峰面积归一法计算每种风味化合物的相对百分含量。
检测结果分析
检测到10种酸类物质，第四天乙酸和正己酸含量高于对照组，是由于乳酸菌发酵过程中产酸；检测到22种醇类物质，前10天乙醇含量高于对照组，是由于乳酸菌发酵过程中，促进糖类的代谢；检测到14种醛类物质，苯甲醛和苯乙醛的相对含量呈现上升趋势但低于对照组，是由于发酵过程中羰基的形成，而添加菌株有利于减缓羰基的生成；随着发酵时间的延长，酯类物质的相对含量升高，共检测到42种酯类物质，其中己酸乙酯具有曲香和菠萝香气，己酸己酯是一种具有清香果味的食用原料，辛酸正丁酯是一种具有黄油香型气味的物质，甲酸芳樟酯具有水果和玫瑰花的香气，(E)-2-甲基-2-丁烯酸己酯是一种合成香料；检测到萜烯类物质高于对照组，是由于乳酸菌的加入促进香辛料中风味物质的挥发。
将该菌株接种于中式发酵香肠中，在发酵过程中形成特殊的风味，GC-MS气质联用共检测到153种挥发性物质，香肠中酯类物质种类由16种增加到20种，酯类的总相对含量增加了17.13％，尤其是己酸乙酯(增加了15.32％)、癸酸乙酯(增加了39.82％)、壬酸乙酯(增加了52.63％)、辛酸乙酯(增加了50％)和(E)-2-甲基-2-丁烯酸己酯(增加了73.62％)等特征风味物质，说明该菌株有助于传统中式香肠风味物质的产生。
将该菌株接种到中式发酵香肠中发现，该菌株的添加能够有效延缓香肠脂肪和蛋白质氧化。能够显著减缓香肠TBARs值，发酵前期显著降低POV值和LOX酶活性(p<0.05)，说明该菌株能够延缓香肠中脂肪氧化；在发酵过程中该菌株还能够抑制香肠肌浆蛋白与肌原纤维蛋白的羰基形成，减缓了其巯基的流失，同时降低了蛋白质二级结构α-螺旋结构相对含量的损失，减少蛋白表面疏水基团的暴露，表明菌株具有良好的延缓香肠中蛋白质氧化的效果。通过检测高铁肌红蛋白的含量，结果表明本发明保藏的乳酸菌能够减少MetMb的生成，降低肌红蛋白被氧化的速度。
所获得的一株植物乳杆菌具有降解亚硝酸盐、抗氧化和产香能力，菌株对酸、牛胆盐、亚硝酸盐及NaCl均具有耐受性；在低浓度NaNO2下，降解率可达到94.85±2.96％，且亚硝酸盐还原酶活力为174.60±13.67U/104cell。根据分析，说明植物乳杆菌FQR可在低浓度NaNO2下高效降解亚硝酸盐，且在酸、牛胆盐、亚硝酸盐及NaCl外界环境刺激下，菌株仍具有降解亚硝酸盐和抗氧化的能力。同时添加乳酸菌制备中式发酵香肠，可以有效抑制脂肪和蛋白氧化，促进风味物质的形成。所述耐受酸、牛胆盐、亚硝酸盐及NaCl的数据见下表。
表1植物乳杆菌FQR对酸的耐受情况
表2植物乳杆菌FQR对胆盐的耐受情况
表3植物乳杆菌FQR对亚硝酸盐的耐受情况
表4植物乳杆菌FQR对NaCl的耐受情况
Claims (4)
1. 一株降解亚硝酸盐、抗氧化和产香的植物乳杆菌，其特征在于：所述植物乳杆菌为植物乳杆菌Lactobacillus plantarum WCFS1，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO: M2021228。
2. 一种权利要求1所述的降解亚硝酸盐、抗氧化和产香的植物乳杆菌的应用，其特征在于：所述植物乳杆菌Lactobacillus plantarum WCFS1用在酸、牛胆盐、亚硝酸盐及NaCl的环境中，并用于降解亚硝酸盐，降低环境中亚硝酸盐含量，同时具有抗氧化作用。
3. 一种权利要求1所述的降解亚硝酸盐、抗氧化和产香的植物乳杆菌的应用，其特征在于：所述植物乳杆菌Lactobacillus plantarum WCFS1用于发酵肉制品的生产中，用于降低肉制品中亚硝酸盐含量，同时延缓脂肪和蛋白的氧化。
4. 一种权利要求1所述的降解亚硝酸盐、抗氧化和产香的植物乳杆菌的应用，其特征在于：所述植物乳杆菌Lactobacillus plantarum WCFS1用于制备香肠，降低香肠腌制过程中亚硝酸盐含量，同时延缓香肠脂肪和蛋白质氧化，并提高香气物质的产生量。
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