Article(id=1320882022680219984, tenantId=1146029695717560320, journalId=1260987677001138203, issueId=1320882022160126288, articleNumber=null, orderNo=null, doi=10.13386/j.issn1002-0306.2024120181, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1734278400000, receivedDateStr=2024-12-16, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1792852415007, onlineDateStr=2026-10-24, pubDate=1767196800000, pubDateStr=2026-01-01, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792852415007, onlineIssueDateStr=2026-10-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792852415007, creator=13701087609, updateTime=1792852415007, updator=13701087609, issue=Issue{id=1320882022160126288, tenantId=1146029695717560320, journalId=1260987677001138203, year='2026', volume='47', issue='1', pageStart='1', pageEnd='498', issueExtLink='null', onlineDate='null', pubDate='1767196800000', pubDateStr='2026-01-01', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792852414884, creator='13701087609', updateTime=1792852414884, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=206, endPage=214, ext={EN=ArticleExt(id=1320882022881546577, articleId=1320882022680219984, tenantId=1146029695717560320, journalId=1260987677001138203, language=EN, title=Screening of High Yielding Antioxidant Peptide Bacillus sphaericus and Its Application in Value-added Utilization of Soybean Residue, columnId=1261336275769016441, journalTitle=Science and Technology of Food Industry, columnName=Bioengineering, runingTitle=null, highlight=null, articleAbstract=

Objective: Soybean residue, a major by-product of soybean processing, possess nutritional value but have low industrial utilization due to low digestibility and poor palatability. The aim of this study was to use soybean residue as a fermentation substrate, take advantage of the characteristics of Bacillus sp. that can grow and multiply in food materials and secrete an extracellular enzyme system, and explore an effective way to prepare small molecule bioactive substances such as antioxidant peptides with the help of ultrasonic-combined microbial fermentation (UAF), thereby enhancing the utilization value of soybean residue. Methods: In this study, 28 high-protease-producing strains were screened from 56 strains of Bacillus sp. by initial screening using the transparent-circle method. Then, their protease activity, superoxide dismutase activity, peptide-producing capacity, and antioxidant capacity were comprehensively evaluated through re-screening in combination with factor analysis. Results: Four strains with antioxidant capacity, namely B. siamensis s4, B. amyloliquefaciens 10, B. subtilis SNBS-3, and B. subtilis 63, were finally screened out. Among them, the protease activity, ABTS+ radical scavenging rate, and DPPH radical scavenging rate of B. amyloliquefaciens 10 were significantly increased (P<0.05) 12.23%±0.08%, 8.69%±0.11%, and 3.38%±0.81%, respectively. Meanwhile, the peptide content and DPPH radical scavenging rate of B. subtilis SNBS-3 were significantly increased (P<0.05) 7.20%±0.10%, 3.41%±0.14%, respectively. Conclusion: Using soybean residue as a fermentation substrate, ultrasonication combined with microbial fermentation could be regarded as a method to screen strains with high antioxidant peptide production. Under ultrasonication stress conditions, the two strains, B. amyloliquefaciens 10 and B. subtilis SNBS-3, not only demonstrated good growth ability but also significantly enhanced protease activity, peptide content, and antioxidant capacity. They had strong antioxidant potential and provided theoretical support for the industrialization of antioxidant peptide preparation.

, authors=Qu LIU1, 2, Feiyu AN1, 2, Rina WU1, 2, 3, Ye TIAN4, Zhen TIAN4, Guoyang PAN1, 3, Jinhui JIANG1, 2, Xinyu TAO1, 3, Yuanyuan MA1, 2, Junrui WU1, 2, 3, *, authorsList=Qu LIU, Feiyu AN, Rina WU, Ye TIAN, Zhen TIAN, Guoyang PAN, Jinhui JIANG, Xinyu TAO, Yuanyuan MA, Junrui WU, authorCompany=null, correspAuthors=Junrui WU, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright © 2026 Science and Technology of Food Industry. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1320882023577801044, articleId=1320882022680219984, tenantId=1146029695717560320, journalId=1260987677001138203, language=CN, title=高产抗氧化肽芽孢杆菌的筛选及其在豆渣增值利用中的应用, columnId=1261336277547401341, journalTitle=食品工业科技, columnName=生物工程, runingTitle=null, highlight=null, articleAbstract=

目的:豆渣作为大豆加工主要副产物,虽有营养价值,但因消化率低、适口性差导致工业利用率低。本研究旨在以豆渣为发酵基质,利用芽孢杆菌在食品原料中生长繁殖并分泌胞外酶系的特性,借助超声波联合微生物发酵方式(UAF),探索制备抗氧化肽等小分子生物活性物的有效途径,提升豆渣的利用价值。方法:通过透明圈法初筛,从56株芽孢杆菌中筛选出28株高产蛋白酶的菌株,并通过复筛对其蛋白酶活力、超氧化物歧化酶活力、产肽能力和抗氧化能力等指标结合因子分析进行综合评价。结果:筛选出B. siamensis s4、B. amyloliquefaciens 10、B. subtilis SNBS-3和B. subtilis 63四株具有抗氧化能力的菌株。其中,B. amyloliquefaciens 10的蛋白酶活性、ABTS+自由基清除率和DPPH自由基清除率分别显著(P<0.05)提高了12.23%±0.08%、8.69%±0.11%和3.38%±0.81%,而B. subtilis SNBS-3的肽含量和DPPH自由基清除率分别显著(P<0.05)提高了7.20%±0.10%、3.41%±0.14%。结论:以豆渣为发酵基质,超声联合微生物发酵方式可作为筛选高产抗氧化肽菌株的方法;在超声胁迫条件下,B. amyloliquefaciens 10和B. subtilis SNBS-3两株菌不仅表现出良好的生长能力,还显著提高了蛋白酶活性、肽含量和抗氧化能力。这两株菌具有强大的抗氧化潜力,为抗氧化肽的产业化制备提供了理论支持。

, authors=刘曲1, 2, 安飞宇1, 2, 乌日娜1, 2, 3, 田野4, 田震4, 潘国杨1, 3, 姜锦惠1, 2, 陶鑫禹1, 3, 马媛媛1, 2, 武俊瑞1, 2, 3, *, authorsList=刘曲, 安飞宇, 乌日娜, 田野, 田震, 潘国杨, 姜锦惠, 陶鑫禹, 马媛媛, 武俊瑞, authorCompany=null, correspAuthors=武俊瑞, authorNote=

刘曲(1999−)(ORCID:0009−0000−0690−0227),女,硕士研究生,研究方向:食品生物技术,E-mail:

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武俊瑞(1977−)(ORCID:0000−0003−3419−0108),男,博士,教授,研究方向:食品生物技术,E-mail:。
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Preparation, purification, and identification of novel feather keratin-derived peptides with antioxidative and xanthine oxidase inhibitory activities[J]. Journal of Agricultural and Food Chemistry, 2023, 71(21): 8061−8070., articleTitle=null, refAbstract=null), Reference(id=1320882038039761380, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=32, rfOrder=36, authorNames=null, journalName=null, refType=null, unstructuredReference=丁学知, 高必达. 高产超氧化物歧化酶芽孢杆菌C328菌株产酶培养基的研究[J]. 湖南师范大学自然科学学报, 2003, 26(4): 65−69. [DING Xuezhi, GAO Bida. Study on medium of highly producing superoxide dismutase from Bacillus cereus strain C328[J]. 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注:不同小写字母表示同一指标测定下不同菌株之间差异显著(P<0.05)。

, figureFileSmall=yR9QT3pOIaAYxSN+Zi52KA==, figureFileBig=1EvQzDdQSTSgOR2t1e+GwQ==, tableContent=null), ArticleFig(id=1320882031412761004, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 1, caption=

Comparison of 16S rDNA sequence homology among strains

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株来源菌株编号鉴定结果同源性(%)
沈阳发酵豆酱5-3B. subtilis99.8
s19B. velezensis99.8
SN-14B. subtilis99.7
5-2B. amyloliquefaciens98.9
31B. amyloliquefaciens99.9
71B. velezensis98.8
J19B. subtilis98.4
63B. subtilis100
17-1B. amyloliquefaciens99.5
21B. velezensis98.8
s16B. subtilis98.4
101B. velezensis99.8
15B. amyloliquefaciens99.8
25B. amyloliquefaciens99.7
26B. amyloliquefaciens99.8
锦州发酵小菜104B. amyloliquefaciens98.8
100B. subtilis98.4
j10B. velezensis99.2
116B. amyloliquefaciens99.8
117B. amyloliquefaciens99.8
105B. amyloliquefaciens99.7
103B. subtilis99.8
6-2B. velezensis99.8
SNBS-3B. subtilis100
辽阳发酵豆豉j21B. subtilis98.4
j11B. subtilis99.2
j14B. velezensis98.4
8"B. subtilis99.2
73B. velezensis99.5
111B. subtilis98.8
10B. amyloliquefaciens100
24"B. amyloliquefaciens99.5
s9B. amyloliquefaciens98.8
朝阳发酵豆酱23B. subtilis98.4
j20B. pseudomycoide99.8
24B. velezensis99.2
j12B. velezensis99.5
s4B. siamensis98.8
7B. velezensis98.4
29B. amyloliquefaciens99.8
阜新发酵豆豉s1B. subtilis99.8
s14B. amyloliquefaciens99.7
s3B. subtilis99.8
43B. subtilis99.5
TSB. subtilis98.8
葫芦岛发酵小菜J20B. velezensis98.4
113 B. amyloliquefaciens99.8
114B. subtilis99.5
112B. amyloliquefaciens98.8
J8B. velezensis100
S29B. amyloliquefaciens100
43B. amyloliquefaciens99.5
J40B. amyloliquefaciens98.8
44B. subtilis98.4
J32B. pseudomycoide99.8
8-3B. velezensis99.2
24B. velezensis99.2
j12B. velezensis99.5
), ArticleFig(id=1320882031488258477, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表1, caption=

菌株16S rDNA序列同源性对比

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株来源菌株编号鉴定结果同源性(%)
沈阳发酵豆酱5-3B. subtilis99.8
s19B. velezensis99.8
SN-14B. subtilis99.7
5-2B. amyloliquefaciens98.9
31B. amyloliquefaciens99.9
71B. velezensis98.8
J19B. subtilis98.4
63B. subtilis100
17-1B. amyloliquefaciens99.5
21B. velezensis98.8
s16B. subtilis98.4
101B. velezensis99.8
15B. amyloliquefaciens99.8
25B. amyloliquefaciens99.7
26B. amyloliquefaciens99.8
锦州发酵小菜104B. amyloliquefaciens98.8
100B. subtilis98.4
j10B. velezensis99.2
116B. amyloliquefaciens99.8
117B. amyloliquefaciens99.8
105B. amyloliquefaciens99.7
103B. subtilis99.8
6-2B. velezensis99.8
SNBS-3B. subtilis100
辽阳发酵豆豉j21B. subtilis98.4
j11B. subtilis99.2
j14B. velezensis98.4
8"B. subtilis99.2
73B. velezensis99.5
111B. subtilis98.8
10B. amyloliquefaciens100
24"B. amyloliquefaciens99.5
s9B. amyloliquefaciens98.8
朝阳发酵豆酱23B. subtilis98.4
j20B. pseudomycoide99.8
24B. velezensis99.2
j12B. velezensis99.5
s4B. siamensis98.8
7B. velezensis98.4
29B. amyloliquefaciens99.8
阜新发酵豆豉s1B. subtilis99.8
s14B. amyloliquefaciens99.7
s3B. subtilis99.8
43B. subtilis99.5
TSB. subtilis98.8
葫芦岛发酵小菜J20B. velezensis98.4
113 B. amyloliquefaciens99.8
114B. subtilis99.5
112B. amyloliquefaciens98.8
J8B. velezensis100
S29B. amyloliquefaciens100
43B. amyloliquefaciens99.5
J40B. amyloliquefaciens98.8
44B. subtilis98.4
J32B. pseudomycoide99.8
8-3B. velezensis99.2
24B. velezensis99.2
j12B. velezensis99.5
), ArticleFig(id=1320882031567950254, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 2, caption=

Preliminary screening test on strain protease production

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号比值(D·d−1)菌株编号比值(D·d−1)菌株编号比值(D·d−1)
108.44±0.06aJ323.67±0.03ijklmnj212.33±0.03p
SNBS-38.32±0.01a433.6±0.06ijklmn8"2.3±0.12pq
637.22±0.14bs163.6±0.01ijklmn6-22.26±0.04pq
s46.00±0.12cj103.57±0.03ijklmnJ201.84±0.01qr
s35.73±0.27c1043.49±0.01ijklmn1121.66±0.01rs
J195.08±0.04d1173.47±0.03jklmns191.58±0.04rs
5-24.83±0.14de1113.4±0.06klmn1031.52±0.03rs
444.47±0.08efS293.33±0.07lmn311.45±0.05rs
244.47±0.08efs93.33±0.07lmnj141.43±0.03rs
254.46±0.08ef1003.21±0.09mn43"1.4±0.06rs
5-34.32±0.03fg263.08±0.03no231.4±0.06rs
8-34.32±0.03fg732.77±0.03op1051.35±0.06rs
SN-144.13±0.04fgh17-12.57±0.07p1141.34±0.01rs
214.02±0.07fghi152.47±0.09p1011.27±0.07s
s143.96±0.07fghij1162.47±0.03pj201.2±0.03s
s13.93±0.05ghijk712.38±0.54pJ401.2±0.03s
TS3.84±0.01ghijklJ82.38±0.06p24"1.19±0.02s
73.84±0.01ghijklj112.37±0.03p291.16±0.01t
j123.67±0.03ijklmn1132.36±0.03p
), ArticleFig(id=1320882031651836335, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表2, caption=

菌株产蛋白酶能力初筛

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号比值(D·d−1)菌株编号比值(D·d−1)菌株编号比值(D·d−1)
108.44±0.06aJ323.67±0.03ijklmnj212.33±0.03p
SNBS-38.32±0.01a433.6±0.06ijklmn8"2.3±0.12pq
637.22±0.14bs163.6±0.01ijklmn6-22.26±0.04pq
s46.00±0.12cj103.57±0.03ijklmnJ201.84±0.01qr
s35.73±0.27c1043.49±0.01ijklmn1121.66±0.01rs
J195.08±0.04d1173.47±0.03jklmns191.58±0.04rs
5-24.83±0.14de1113.4±0.06klmn1031.52±0.03rs
444.47±0.08efS293.33±0.07lmn311.45±0.05rs
244.47±0.08efs93.33±0.07lmnj141.43±0.03rs
254.46±0.08ef1003.21±0.09mn43"1.4±0.06rs
5-34.32±0.03fg263.08±0.03no231.4±0.06rs
8-34.32±0.03fg732.77±0.03op1051.35±0.06rs
SN-144.13±0.04fgh17-12.57±0.07p1141.34±0.01rs
214.02±0.07fghi152.47±0.09p1011.27±0.07s
s143.96±0.07fghij1162.47±0.03pj201.2±0.03s
s13.93±0.05ghijk712.38±0.54pJ401.2±0.03s
TS3.84±0.01ghijklJ82.38±0.06p24"1.19±0.02s
73.84±0.01ghijklj112.37±0.03p291.16±0.01t
j123.67±0.03ijklmn1132.36±0.03p
), ArticleFig(id=1320882031723139504, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 3, caption=

Protease activity in fermented soybean residue peptide solution

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号蛋白酶活力(U·mL−1)菌株编号蛋白酶活力(U·mL−1)
s4566.29±1.59a117275.30±0.86q
J19498.60±2.90d104247.04±0.44r
SN-14332.69±1.57n111405.15±0.90j
5-2482.88±3.25e5-3408.46±0.52i
S29306.84±0.35ps9354.98±1.14m
10566.05±0.57a24350.59±0.13m
63565.92±1.50aj12438.90±0.94h
21469.71±1.72f8-3392.52±0.90k
SNBS-3556.61±4.67b7388.57±0.65k
25243.94±1.10rs1486.87±0.91e
44508.83±1.00cs14456.31±2.04g
s16412.20±0.81is3469.02±0.64f
J32322.32±0.78o43434.19±0.70h
j10380.79±0.79lTS272.46±1.21q
), ArticleFig(id=1320882031807025585, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表3, caption=

菌株发酵豆渣多肽液蛋白酶活力

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号蛋白酶活力(U·mL−1)菌株编号蛋白酶活力(U·mL−1)
s4566.29±1.59a117275.30±0.86q
J19498.60±2.90d104247.04±0.44r
SN-14332.69±1.57n111405.15±0.90j
5-2482.88±3.25e5-3408.46±0.52i
S29306.84±0.35ps9354.98±1.14m
10566.05±0.57a24350.59±0.13m
63565.92±1.50aj12438.90±0.94h
21469.71±1.72f8-3392.52±0.90k
SNBS-3556.61±4.67b7388.57±0.65k
25243.94±1.10rs1486.87±0.91e
44508.83±1.00cs14456.31±2.04g
s16412.20±0.81is3469.02±0.64f
J32322.32±0.78o43434.19±0.70h
j10380.79±0.79lTS272.46±1.21q
), ArticleFig(id=1320882031911883186, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 4, caption=

Peptide content in fermented soybean residue peptide solution

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号肽含量(mg·mL−1)菌株编号肽含量(mg·mL−1)
s48.74±0.05b1174.79±0.01q
J197.73±0.04e1044.14±0.10r
SN-145.65±0.04n1116.57±0.04k
5-27.43±0.09fg5-36.57±0.07k
S295.03±0.03ps95.99±0.01m
108.50±0.07c245.86±0.04m
639.06±0.13aj126.74±0.02j
217.30±0.10gh8-36.34±0.06l
SNBS-38.76±0.09b76.26±0.02l
253.96±0.03ss17.49±0.11fg
448.10±0.04ds146.96±0.03i
s166.58±0.02ks37.24±0.03h
J325.37±0.03o436.70±0.03jk
j106.27±0.02lTS4.68±0.02q
), ArticleFig(id=1320882031987380659, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表4, caption=

菌株发酵豆渣多肽液多肽含量

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号肽含量(mg·mL−1)菌株编号肽含量(mg·mL−1)
s48.74±0.05b1174.79±0.01q
J197.73±0.04e1044.14±0.10r
SN-145.65±0.04n1116.57±0.04k
5-27.43±0.09fg5-36.57±0.07k
S295.03±0.03ps95.99±0.01m
108.50±0.07c245.86±0.04m
639.06±0.13aj126.74±0.02j
217.30±0.10gh8-36.34±0.06l
SNBS-38.76±0.09b76.26±0.02l
253.96±0.03ss17.49±0.11fg
448.10±0.04ds146.96±0.03i
s166.58±0.02ks37.24±0.03h
J325.37±0.03o436.70±0.03jk
j106.27±0.02lTS4.68±0.02q
), ArticleFig(id=1320882032058683828, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 5, caption=

Antioxidant capacity of peptide solution from fermented soybean residue

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号ABTS+自由基清除率(%)DPPH自由基清除率(%)SOD酶活力
(U·mL−1)
菌株编号ABTS+自由基清除率(%)DPPH自由基清除率(%)SOD酶活力
(U·mL−1)
s464.34±0.8b44.46±0.52b365.93±2.34b11732.94±0.32m21.49±0.23o200.42±0.14p
J1956.41±0.77c38.43±0.59d307.01±0.60d10429.14±0.31n21.58±0.26o172.76±2.02q
SN-1440.67±0.22j23.97±0.14m229.50±0.81n11150.44±0.53ef30.28±0.34i249.43±0.71jk
5-252.91±1.09d37.06±0.70e298.76±1.65e5-350.88±0.29e30.57±0.19i251.81±0.45ij
S2937.19±0.12l21.71±0.08o217.03±0.51os943.67±0.16i25.91±0.10l236.72±0.39m
1064.72±0.52b44.70±0.33b368.00±1.4bc2443.09±0.2i25.53±0.12l234.07±0.71mn
6363.4±1.28b43.84±0.83b371.53±0.96aj1254.98±0.51c33.22±0.33h267.86±0.20h
2151.13±0.34e35.91±0.22f288.93±1.40f8-348.74±0.35g29.18±0.23j249.46±1.24jk
SNBS-368.94±0.45a45.62±0.28a368.31±2.39b748.2±0.36gh28.84±0.24jk245.23±0.69kl
2528.72±0.24n21.23±0.20o166.74±0.30rs153.45±0.84d37.41±0.55e305.40±0.97d
4455.03±0.91c39.32±0.50c312.25±0.08cs1449.33±0.7fg34.74±0.45g277.14±1.25g
s1643.39±0.31i30.90±0.20i255.58±0.23is351.05±0.7e35.85±0.45f289.60±0.98f
J3239.28±0.29k23.07±0.19n221.00±0.80o4346.35±0.42h32.82±0.27h265.05±0.69h
j1047.16±0.33h28.16±0.22k242.47±0.27lTS32.56±0.13m21.21±0.10o201.21±1.36p
), ArticleFig(id=1320882032129986997, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表5, caption=

菌株发酵豆渣多肽液抗氧化能力

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号ABTS+自由基清除率(%)DPPH自由基清除率(%)SOD酶活力
(U·mL−1)
菌株编号ABTS+自由基清除率(%)DPPH自由基清除率(%)SOD酶活力
(U·mL−1)
s464.34±0.8b44.46±0.52b365.93±2.34b11732.94±0.32m21.49±0.23o200.42±0.14p
J1956.41±0.77c38.43±0.59d307.01±0.60d10429.14±0.31n21.58±0.26o172.76±2.02q
SN-1440.67±0.22j23.97±0.14m229.50±0.81n11150.44±0.53ef30.28±0.34i249.43±0.71jk
5-252.91±1.09d37.06±0.70e298.76±1.65e5-350.88±0.29e30.57±0.19i251.81±0.45ij
S2937.19±0.12l21.71±0.08o217.03±0.51os943.67±0.16i25.91±0.10l236.72±0.39m
1064.72±0.52b44.70±0.33b368.00±1.4bc2443.09±0.2i25.53±0.12l234.07±0.71mn
6363.4±1.28b43.84±0.83b371.53±0.96aj1254.98±0.51c33.22±0.33h267.86±0.20h
2151.13±0.34e35.91±0.22f288.93±1.40f8-348.74±0.35g29.18±0.23j249.46±1.24jk
SNBS-368.94±0.45a45.62±0.28a368.31±2.39b748.2±0.36gh28.84±0.24jk245.23±0.69kl
2528.72±0.24n21.23±0.20o166.74±0.30rs153.45±0.84d37.41±0.55e305.40±0.97d
4455.03±0.91c39.32±0.50c312.25±0.08cs1449.33±0.7fg34.74±0.45g277.14±1.25g
s1643.39±0.31i30.90±0.20i255.58±0.23is351.05±0.7e35.85±0.45f289.60±0.98f
J3239.28±0.29k23.07±0.19n221.00±0.80o4346.35±0.42h32.82±0.27h265.05±0.69h
j1047.16±0.33h28.16±0.22k242.47±0.27lTS32.56±0.13m21.21±0.10o201.21±1.36p
), ArticleFig(id=1320882032209678774, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 6, caption=

Results of KMO and Bartlett test

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指标结果
取样足够度的KMO度量0.844
Bartlett的球形度检验均卡方1173.241
df10
Sig.0.0001
), ArticleFig(id=1320882032276787639, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表6, caption=

KMO和Bartlett球形检验

, figureFileSmall=null, figureFileBig=null, tableContent=
指标结果
取样足够度的KMO度量0.844
Bartlett的球形度检验均卡方1173.241
df10
Sig.0.0001
), ArticleFig(id=1320882032339702200, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 7, caption=

Eigenvalues and variance contributions

, figureFileSmall=null, figureFileBig=null, tableContent=
成份初始特征值提取载荷平方和旋转载荷平方和
总计方差百分比(%)累积
(%)
总计方差百分比(%)累积
(%)
总计方差百分比(%)累积
(%)
14.90097.99497.9944.90097.99497.9942.70754.14954.149
20.0541.07899.0730.0541.07899.0732.24644.92499.073
30.0240.48999.562
40.0180.35799.919
50.0040.081100.000
), ArticleFig(id=1320882032406811065, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表7, caption=

特征值和方差贡献率

, figureFileSmall=null, figureFileBig=null, tableContent=
成份初始特征值提取载荷平方和旋转载荷平方和
总计方差百分比(%)累积
(%)
总计方差百分比(%)累积
(%)
总计方差百分比(%)累积
(%)
14.90097.99497.9944.90097.99497.9942.70754.14954.149
20.0541.07899.0730.0541.07899.0732.24644.92499.073
30.0240.48999.562
40.0180.35799.919
50.0040.081100.000
), ArticleFig(id=1320882032465531322, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 8, caption=

Score coefficient matrix

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因子12
X1蛋白酶活性0.462−0.206
X2超氧化物歧化酶活性0.854−0.639
X3肽含量0.0250.274
X4 ABTS+自由基清除率−2.2272.747
X5 DPPH自由基清除率1.612−1.474
), ArticleFig(id=1320882032541028795, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表8, caption=

得分系数矩阵

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因子12
X1蛋白酶活性0.462−0.206
X2超氧化物歧化酶活性0.854−0.639
X3肽含量0.0250.274
X4 ABTS+自由基清除率−2.2272.747
X5 DPPH自由基清除率1.612−1.474
), ArticleFig(id=1320882032612331964, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=EN, label=Table 9, caption=

Component composite scores

, figureFileSmall=null, figureFileBig=null, tableContent=
菌株编号F1F2因子综合评分F排序菌株编号F1F2因子综合评分F排序
s41.350.971.551s140.71−0.360.0915
101.350.951.5327−1.090.820.0916
631.520.831.533j10−1.080.720.0117
SNBS-31.530.811.524430.67−0.67−0.1918
J190.600.580.815s9−1.000.27−0.3219
j12−1.071.560.71624−1.010.19−0.3920
441.220.020.77s160.67−0.97−0.4421
s10.95−0.030.58SN-14−0.96−0.11−0.6222
520.87−0.040.459J32−1.03−0.25−0.7823
53−1.111.160.3610S29−0.99−0.55−1.0124
111−1.131.130.3311117−0.51−1.45−1.4925
210.81−0.190.2912TS−0.61−1.33−1.5726
s30.82−0.220.2713104−0.60−1.39−2.0127
8-3−1.050.860.141425−0.32−2.26−2.0628
), ArticleFig(id=1320882032696218045, tenantId=1146029695717560320, journalId=1260987677001138203, articleId=1320882022680219984, language=CN, label=表9, caption=

综合评分表

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菌株编号F1F2因子综合评分F排序菌株编号F1F2因子综合评分F排序
s41.350.971.551s140.71−0.360.0915
101.350.951.5327−1.090.820.0916
631.520.831.533j10−1.080.720.0117
SNBS-31.530.811.524430.67−0.67−0.1918
J190.600.580.815s9−1.000.27−0.3219
j12−1.071.560.71624−1.010.19−0.3920
441.220.020.77s160.67−0.97−0.4421
s10.95−0.030.58SN-14−0.96−0.11−0.6222
520.87−0.040.459J32−1.03−0.25−0.7823
53−1.111.160.3610S29−0.99−0.55−1.0124
111−1.131.130.3311117−0.51−1.45−1.4925
210.81−0.190.2912TS−0.61−1.33−1.5726
s30.82−0.220.2713104−0.60−1.39−2.0127
8-3−1.050.860.141425−0.32−2.26−2.0628
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高产抗氧化肽芽孢杆菌的筛选及其在豆渣增值利用中的应用
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刘曲 1, 2 , 安飞宇 1, 2 , 乌日娜 1, 2, 3 , 田野 4 , 田震 4 , 潘国杨 1, 3 , 姜锦惠 1, 2 , 陶鑫禹 1, 3 , 马媛媛 1, 2 , 武俊瑞 1, 2, 3, *
食品工业科技 | 生物工程 2026,47(1): 206-214
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食品工业科技 |生物工程 2026 , 47 (1) : 206 -214
高产抗氧化肽芽孢杆菌的筛选及其在豆渣增值利用中的应用
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刘曲1, 2 , 安飞宇1, 2, 乌日娜1, 2, 3, 田野4, 田震4, 潘国杨1, 3, 姜锦惠1, 2, 陶鑫禹1, 3, 马媛媛1, 2, 武俊瑞1, 2, 3, *
作者信息
  • 1沈阳农业大学食品学院,辽宁沈阳 110866
  • 2辽宁省食品发酵技术工程研究中心,辽宁沈阳 110866
  • 3沈阳市微生物发酵技术创新重点实验室,辽宁沈阳 110866
  • 4辽宁帝华味精食品有限公司,辽宁葫芦岛 125200
通讯作者:
武俊瑞(1977−)(ORCID:0000−0003−3419−0108),男,博士,教授,研究方向:食品生物技术,E-mail:。
作者简介:

刘曲(1999−)(ORCID:0009−0000−0690−0227),女,硕士研究生,研究方向:食品生物技术,E-mail:

Screening of High Yielding Antioxidant Peptide Bacillus sphaericus and Its Application in Value-added Utilization of Soybean Residue
Qu LIU1, 2 , Feiyu AN1, 2, Rina WU1, 2, 3, Ye TIAN4, Zhen TIAN4, Guoyang PAN1, 3, Jinhui JIANG1, 2, Xinyu TAO1, 3, Yuanyuan MA1, 2, Junrui WU1, 2, 3, *
Affiliations
  • 1College of Food Science, Shenyang Agricultural University, Shenyang 110866, China
  • 2Liaoning Engineering Research Center of Food Fermentation Technology, Shenyang 110866, China
  • 3Shenyang Key Laboratory of Microbial Fermentation Technology Innovation, Shenyang 110866, China
  • 4Liaoning Dihua Monosodium Glutamate Food Co., Ltd., Huludao 125200, China
出版时间: 2026-01-01 doi: 10.13386/j.issn1002-0306.2024120181
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目的:豆渣作为大豆加工主要副产物,虽有营养价值,但因消化率低、适口性差导致工业利用率低。本研究旨在以豆渣为发酵基质,利用芽孢杆菌在食品原料中生长繁殖并分泌胞外酶系的特性,借助超声波联合微生物发酵方式(UAF),探索制备抗氧化肽等小分子生物活性物的有效途径,提升豆渣的利用价值。方法:通过透明圈法初筛,从56株芽孢杆菌中筛选出28株高产蛋白酶的菌株,并通过复筛对其蛋白酶活力、超氧化物歧化酶活力、产肽能力和抗氧化能力等指标结合因子分析进行综合评价。结果:筛选出B. siamensis s4、B. amyloliquefaciens 10、B. subtilis SNBS-3和B. subtilis 63四株具有抗氧化能力的菌株。其中,B. amyloliquefaciens 10的蛋白酶活性、ABTS+自由基清除率和DPPH自由基清除率分别显著(P<0.05)提高了12.23%±0.08%、8.69%±0.11%和3.38%±0.81%,而B. subtilis SNBS-3的肽含量和DPPH自由基清除率分别显著(P<0.05)提高了7.20%±0.10%、3.41%±0.14%。结论:以豆渣为发酵基质,超声联合微生物发酵方式可作为筛选高产抗氧化肽菌株的方法;在超声胁迫条件下,B. amyloliquefaciens 10和B. subtilis SNBS-3两株菌不仅表现出良好的生长能力,还显著提高了蛋白酶活性、肽含量和抗氧化能力。这两株菌具有强大的抗氧化潜力,为抗氧化肽的产业化制备提供了理论支持。

抗氧化肽  /  蛋白酶  /  芽孢杆菌  /  超声波处理  /  微生物发酵  /  豆渣

Objective: Soybean residue, a major by-product of soybean processing, possess nutritional value but have low industrial utilization due to low digestibility and poor palatability. The aim of this study was to use soybean residue as a fermentation substrate, take advantage of the characteristics of Bacillus sp. that can grow and multiply in food materials and secrete an extracellular enzyme system, and explore an effective way to prepare small molecule bioactive substances such as antioxidant peptides with the help of ultrasonic-combined microbial fermentation (UAF), thereby enhancing the utilization value of soybean residue. Methods: In this study, 28 high-protease-producing strains were screened from 56 strains of Bacillus sp. by initial screening using the transparent-circle method. Then, their protease activity, superoxide dismutase activity, peptide-producing capacity, and antioxidant capacity were comprehensively evaluated through re-screening in combination with factor analysis. Results: Four strains with antioxidant capacity, namely B. siamensis s4, B. amyloliquefaciens 10, B. subtilis SNBS-3, and B. subtilis 63, were finally screened out. Among them, the protease activity, ABTS+ radical scavenging rate, and DPPH radical scavenging rate of B. amyloliquefaciens 10 were significantly increased (P<0.05) 12.23%±0.08%, 8.69%±0.11%, and 3.38%±0.81%, respectively. Meanwhile, the peptide content and DPPH radical scavenging rate of B. subtilis SNBS-3 were significantly increased (P<0.05) 7.20%±0.10%, 3.41%±0.14%, respectively. Conclusion: Using soybean residue as a fermentation substrate, ultrasonication combined with microbial fermentation could be regarded as a method to screen strains with high antioxidant peptide production. Under ultrasonication stress conditions, the two strains, B. amyloliquefaciens 10 and B. subtilis SNBS-3, not only demonstrated good growth ability but also significantly enhanced protease activity, peptide content, and antioxidant capacity. They had strong antioxidant potential and provided theoretical support for the industrialization of antioxidant peptide preparation.

antioxidant peptides  /  proteases  /  Bacillus  /  ultrasonic treatment  /  microbial fermentation  /  soybean residue
刘曲, 安飞宇, 乌日娜, 田野, 田震, 潘国杨, 姜锦惠, 陶鑫禹, 马媛媛, 武俊瑞. 高产抗氧化肽芽孢杆菌的筛选及其在豆渣增值利用中的应用. 食品工业科技, 2026 , 47 (1) : 206 -214 . DOI: 10.13386/j.issn1002-0306.2024120181
Qu LIU, Feiyu AN, Rina WU, Ye TIAN, Zhen TIAN, Guoyang PAN, Jinhui JIANG, Xinyu TAO, Yuanyuan MA, Junrui WU. Screening of High Yielding Antioxidant Peptide Bacillus sphaericus and Its Application in Value-added Utilization of Soybean Residue[J]. Science and Technology of Food Industry, 2026 , 47 (1) : 206 -214 . DOI: 10.13386/j.issn1002-0306.2024120181
豆渣是大豆加工过程中主要的副产物,每加工1 kg大豆就会产生约1.2 kg湿豆渣[1]。我国作为大豆主要消费国之一,每年可产生数亿吨豆渣[2],且预测未来豆渣产量仍会呈逐年递增趋势[3],豆渣的累积已然成为豆制品行业所面临的挑战之一。Chatterjee等[4]研究虽已表明,利用豆渣蛋白质制备生物活性物具有可行性,但残留蛋白质经高温蒸煮、挤压后难以溶出、利用率不佳[5−6]。为此,亟需利用物性修饰、生物转化、功能化重组等副产物综合利用技术以解决上述问题。
生物活性肽是指具有生物活性的小分子肽段。它们通常由2到50个氨基酸残基组成,具有特定的生物功能,如调节免疫系统、细胞增殖、细胞凋亡、细胞信号传导等。生物活性肽的制备方法主要包括微生物发酵、酶解、化学合成与DNA重组技术[7]。其中抗氧化肽是一种具有抗氧化能力的生物活性肽,是一种小分子天然活性物质[8−9]。利用微生物发酵法制备生物活性肽具有安全可靠、多样性、高产量、易于改造、经济性和可持续性等优点,使其成为制备生物活性肽的重要来源之一[10]。目前,已开展多项利用微生物发酵法制备抗氧化肽的研究,如Shu等[11]以羊奶为发酵基质,利用解淀粉芽孢杆菌(B. amyloliquefaciens)制备抗氧化肽;Li等[12]以鹰嘴豆为发酵基质,利用枯草芽孢杆菌(B. subtilis)发酵制备抗氧化肽;Sanjukta等[13]利用枯草芽孢杆菌(B. subtilis)、地衣芽孢杆菌(Bacillus licheniformis)发酵Kinema,制备的生物活性肽SEDDVFVIPAAYPF显示出高抗氧化活性。同时,环保型低强度短时超声波近年来已广泛应用于发酵工业,超声波联合微生物发酵方式(Ultrasonic-combined microbial fermentation,UAF),不仅能促使发酵基质蛋白质结构的舒展,其声空化作用所形成的微射流[14−15],可以提升细胞膜的流动性及渗透性[16],刺激微生物生长、提高蛋白酶产率,还可增强传质速率,提高酶与底物的结合速率及活性成分的溶出率,具有提取效率高、溶剂使用少等优点[17−18],已在食品工业中广泛研究。如Hashemi等[19]利用超声联合发酵,以酸奶为发酵基质,证实通过超声诱导微生物蛋白酶表达可以提高肽的产量;Bian等[20]通过多频逆流超声处理提高了桑籽蛋白提取率,提高了甜味肽的生物活性。因此,微生物发酵法在制备生物活性肽方面优势突出,且超声波联合微生物发酵方式能进一步提升发酵效果,在食品等领域展现出良好的应用潜力。目前,利用豆渣蛋白制备抗氧化肽等小分子活性物的研究较为鲜见,已开展的研究也多采用酶解法、微生物发酵法等单一制备方式;其中,通过超声等辅助处理优化微生物多种代谢途径以发酵制备抗氧化肽的研究,仍处于起步阶段,因此采用超声联合微生物发酵技术制备豆渣抗氧化肽,对实现活性肽的安全高效制备具有重要意义。研究发现,芽孢杆菌(Bacillus)不仅产酶能力较强,是发酵过程中的酶系主导功能菌[21],且菌株活性高,可在豆渣等食品原料上良好生长繁殖[22],但当前针对该菌产抗氧化肽的筛选体系仍存在不完善之处。
因此,本研究通过透明圈法初筛,蛋白酶活力、超氧化物歧化酶活力、多肽含量和抗氧化能力复筛,综合筛选出具有高产抗氧化肽能力的特异性菌株。进一步选用超声波联合微生物发酵法制备抗氧化肽,在提高豆渣利用率的同时赋予经济价值,对提升豆制品副产物利用率,低成本、高安全、高效率制备活性肽具有重要意义,后期为活性肽产业化制备提供理论支持[23]。
菌株 均分离自东北传统发酵食品,菌株16S rDNA序列同源性对比见表1;新鲜豆渣 辽宁豆华天宝食品科技股份有限公司提供;β-巯基乙醇、邻苯二甲醛(OPA)、2,2-联氨-双(3-乙基-苯并噻唑-6-磺酸)二胺盐(ABTS)、1,1-二苯基-2-三硝基苯肼(DPPH)、乙醇、过硫酸钾、甲醇、十水合四硼酸钠(Na2B4O7·10H2O)、十二烷基硫酸钠(SDS) 分析纯,上海麦克林生化科技有限公司;微生物蛋白酶活性检测试剂盒(ELISA)、SOD活性检测试剂盒(ELISA) 北京鼎国昌盛生物技术有限责任公司。
QYC-200恒温培养摇床 上海福玛试验设备有限公司;UV-2100紫外可见分光光度计 龙尼柯(上海)仪器有限公司;KQ-300VDE超声波清洗机 昆山市超声仪器有限公司;DHG-9023S鼓风干燥箱 宁波乐电仪器制造有限公司。
LB(Luria-Bertani)液体培养基、酪蛋白固体培养基:参考Kasana等[24]配制,121 ℃灭菌20 min待用。
豆渣培养基:鲜豆渣于60 ℃鼓风干燥箱中烘干至恒重,10 g干豆渣,水150 mL,121 ℃灭菌20 min待用。
OPA溶液:25 mL蒸馏水溶解0.96 g Na2B4O7·10H2O,2.5 mL蒸馏水溶解0.5 g SDS,1 mL甲醇溶解0.04 g OPA后加0.1 mL β-巯基乙醇,定容至50 mL。
DPPH溶液:100 mL甲醇溶解6.3 mg DPPH。
ABTS溶液:2 mL蒸馏水溶解7.68 mg ABTS、4 mL 蒸馏水溶解2.65 mg K2S2O8,混匀后于室温(25±1 ℃)下避光12~16 h后再溶于无水乙醇,734 nm处调整至吸光度为0.7±0.02待用。
采用透明圈法,将保藏的芽孢杆菌活化后接至LB液体培养基,37 ℃、170 r/min培养16 h后5000 r/min离心10 min弃去上清,0.9%无菌生理盐水调至OD600为0.5±0.02的菌悬液待用。参考Kasana等[24]方法并稍作修改,100 μL菌悬液点种至酪蛋白固体培养基,37 ℃静置培养12 h后用游标卡尺测量透明圈直径(D)及菌落直径(d)。
取1.2.2菌悬液,按1%(v/v)接种至无菌豆渣培养基,37 ℃、170 r/min下培养36 h,取发酵液10000 r/min离心25 min后取上清液过0.45 μm滤膜置于−80 ℃备用。
取多肽液在频率45 kHz、功率120 W/L下超声15 min,使用微生物蛋白酶活性检测试剂盒(ELISA),测定450 nm处吸光度。同时以未超声处理的发酵液为空白组,超声后菌株蛋白酶活力增率根据公式(1)计算。
$ 蛋白酶活力增率({\text{%}})= \frac{{\text{P}}_{{超声}}-{\text{P}}_{{未超声}}}{{\text{P}}_{{未超声}}}\times{100} $
式中:P超声为超声后菌株发酵豆渣多肽液吸光度值;P未超声为未超声菌株发酵豆渣多肽液吸光度值。
参考Tuly等[25]的方法并稍作修改,75 μL 10 mg/mL多肽液与3 mL OPA溶液混匀,室温孵育2 min,测定340 nm处吸光度;配制0、0.2、0.4、0.6、0.8及1.0 mg/mL的Gly-Gly-Tyr-Arg溶液制作标准曲线(Y=11.175X−0.0075,R2=0.9996)。未超声组为空白,超声后菌株肽含量增率根据公式(2)计算。
$ 肽含量增率({\text{%}})= \frac{{\text{C}}_{{超声}}-{\text{C}}_{{未超声}}}{{\text{C}}_{{未超声}}}\times{100} $
式中:C超声为超声后菌株肽含量(mg/mL);C未超声为未超声菌株肽含量(mg/mL)。
取多肽液使用SOD活性检测试剂盒(ELISA),测定波长450 nm处吸光度,按细菌数量计,每1万个细菌每分钟催化产生1 μmol超氧阴离子自由基定义为一个酶活性单位计算样品活性。
参照Rai等[26]的方法并稍作修改。200 μL多肽液与2 mL DPPH溶液混匀,室温下避光孵育30 min,0.2 mg/mL VC为阳性对照,测定517 nm处吸光度,根据公式(3)计算DPPH自由基清除率。
$ {\mathrm{DPPH}}自由基清除率({\text{%}})= \left({1-}\frac{{\mathrm{A}}_{2}-{\mathrm{A}}_{1}}{{\mathrm{A}}_{0}}\right)\times {100} $
式中:A2为样品吸光度值;A1为甲醇溶液代替DPPH溶液吸光度值;A0为去离子水代替样品吸光度值。
此外,未超声组为空白,超声后菌株DPPH自由基清除率增率根据公式(4)计算。
$ {\mathrm{DPPH}}自由基清除率增率({\text{%}})= \frac{{\text{D}}_{1}-{\text{D}}_{0}}{{\text{D}}_{0}}\times{100} $
式中:D1为超声后菌株DPPH自由基清除率;D0为未超声时菌株DPPH自由基清除率。
参照Fang等[27]的方法并稍作修改。1 mL多肽液与4 mL新制ABTS溶液充分混匀,室温下避光孵育6 min,0.2 mg/mL VC为阳性对照,测定波长734 nm处吸光度,根据公式(5)计算ABTS+自由基清除率。
$ {\mathrm{ABTS}}^{+}自由基清除率({\text{%}})= \left({1-}\frac{{\text{A}}_{\text{s}}}{{\text{A}}_{\text{c}}}\right)\times 100 $
式中:As为多肽样品吸光度值;Ac为ABTS溶液空白吸光度值。
此外,未超声组为空白,超声后菌株ABTS+自由基清除率增率根据公式(6)计算。
$ {\mathrm{ABTS}}^{+}自由基清除率增率({\text{%}})= \frac{{\mathrm{A}}_{\mathrm{a}}-{\text{A}}_{\mathrm{b}}}{{\text{A}}_{\mathrm{b}}}\times{100} $
式中:Aa为超声后菌株ABTS+自由基清除率;Ab为未超声时菌株ABTS+自由基清除率。
参考赵自阳等[28]的方法。以28株芽孢杆菌的蛋白酶活性、超氧化物歧化酶活性、肽含量、ABTS+自由基清除率、DPPH自由基清除率为变量,综合评价不同芽孢杆菌的抗氧化能力,计算芽孢杆菌抗氧化能力相关系数矩阵以及相关系数矩阵的特征值和贡献率等,并对因子载荷矩阵进行旋转处理;最终计算芽孢杆菌抗氧化能力的因子综合得分。
将具有发酵潜力的芽孢杆菌,接种1%(v/v)至豆渣培养基,在37 ℃、170 r/min条件下培养4 h后在频率45 kHz、功率120 W/L下超声10 min,再培养36 h,同时以未超声处理的发酵液为空白组,考察超声胁迫对菌株产肽能力、蛋白酶活力、DPPH自由基清除率和ABTS+自由基清除率的影响。
每组实验数据均各测定3次,结果表示为平均值±标准差,采用SPSS软件进行因子分析及样品间显著性差异分析,以P<0.05为差异显著,用Origin 2021、Design-Expert.V8.0.6.1软件绘图。
通过透明圈法对分离自东北传统发酵小菜、发酵豆酱、发酵豆豉的56株芽孢杆菌进行初筛,以优选具有产蛋白酶能力的菌株。结果如表2所示,56株芽孢杆菌均具有产蛋白酶潜力,透明圈的大小直观反映了菌落对蛋白酶的分解能力,透明圈比值(D·d−1)越大产蛋白酶能力越强,56株菌的D·d−1值在1.16±0.01~8.44±0.06之间,平均值为3.22±0.04,通过显著性分析可知,不同菌株间产蛋白酶能力存在显著差异(P<0.05)。其中有28株菌透明圈比值高于平均值,选取前28株具有产蛋白酶潜力的芽孢杆菌进行后续实验。其中B. siamensis s4、B. amyloliquefaciens 10、B. subtilis SNBS-3、B. subtilis 63、B. subtilis s3比值均高于5.50,在产蛋白酶能力方面显著强于其他菌株(P<0.05),表明它们具有较强产蛋白酶潜力。
通过对28株菌的蛋白酶活力进行测定,筛选出具有高产抗氧化肽潜力的菌株。结果如表3所示,28株芽孢杆菌均表现出较强的产酶能力,实验菌株间蛋白酶活力存在显著差异(P<0.05)。其中,B. siamensis s4、B. amyloliquefaciens 10、B. subtilis 63的蛋白酶活力分别为566.29±1.59、566.05±0.57、565.92±1.50 U/mL,且在蛋白酶活力方面处于同一显著水平,表明产蛋白酶能力显著强于其他大部分菌株(P<0.05),产蛋白酶能力较强;B. subtilis s16、B. subtilis 5-3产蛋白酶能力适中,分别为412.20±0.81、408.46±0.52 U/mL,这两株菌的蛋白酶活力水平无显著差异;B. amyloliquefaciens 104、B. amyloliquefaciens 25蛋白酶活力分别为247.04±0.44、243.94±1.10 U/mL,二者蛋白酶活力无显著差异,产酶能力较弱,与前述的透明圈初筛结果相似,可验证上述实验结果。
通过对28株菌的产肽能力进行测定,筛选出产多肽能力强的菌株。结果如表4所示,28株芽孢杆菌均具有产肽的能力,实验菌株间产肽能力存在显著差异(P<0.05)。其中B. subtilis 63肽含量为9.06±0.13 mg/mL,表明产肽能力显著强于其他大部分菌株(P<0.05);B. subtilis 111、B. subtilis 5-3肽含量为6.57±0.04、6.57±0.07 mg/mL,二者蛋白酶活力无显著差异,产肽能力适中,与产肽能力较强的B. subtilis 63相比,具有显著差异(P<0.05);B. amyloliquefaciens 104产肽能力较差,仅为4.14±0.10 mg/mL。通过对28株菌株的蛋白酶活力以及多肽含量测定可以验证,蛋白酶活力可以对多肽含量产生一定影响。大多数菌株蛋白酶活力高,其菌株的产肽能力强。
SOD是一种抗氧化金属酶,在抗氧化平衡中起到至关重要的作用,是生物体系中抗氧化酶系的重要成员之一[29]。抗氧化肽可避免或减轻底物与自由基结合造成的氧化损伤,从而达到抗氧化作用[30]。
已有研究表明,B. amyloliquefaciens、B. subtilis等芽孢杆菌可提升发酵液抗氧化活性,在制备抗氧化生物活性物方面具有一定应用潜力[31]。为此,针对上述28株优产蛋白酶的芽孢杆菌,进一步筛选具有高产SOD酶特性的特异性菌株,结果如表5所示。28株芽孢杆菌均可产SOD酶,实验菌株间酶活力存在显著差异(P<0.05)。其中B. amyloliquefaciens 10、B. subtilis 63 SOD酶活力较强,分别为368.00±1.4、371.53±0.96 U/mL,产酶能力略高于丁学知等[32]筛选出的高产SOD酶的芽孢杆菌C328(358 U/mL)。B. subtilis 43、B. velezensis j12 SOD酶活力分别为265.05±0.69、267.68±0.20 U/mL,二者无显著差异,产酶能力适中,与产酶能力较强的菌株存在显著差异(P<0.05),但又显著高于产酶能力较弱的菌株(P<0.05);B. amyloliquefaciens 25 SOD酶活力为166.74±0.30 U/mL,产酶能力较弱。28株芽孢杆菌的多肽液均具有抗氧化活性,且存在显著差异(P<0.05)。其中B. subtilis SNBS-3多肽液的ABTS+自由基清除率、DPPH自由基清除率分别为68.94%±0.45%、45.62%±0.28%,处于最高水平,表明该菌株的多肽液抗氧化能力显著强于其他大部分菌株(P<0.05);B. amyloliquefaciens 25、B. amyloliquefaciens 104 多肽液ABTS+自由基清除率较差,为28.72%±0.24%、29.14%±0.31%;B. amyloliquefaciens 117、B. amyloliquefaciens 104、B. amyloliquefaciens S29、B. subtilis TS、B. amyloliquefaciens 25 DPPH自由基清除率较低,分别为21.49%±0.23%、21.58%±0.26%、21.71%±0.08%、21.21%±0.10%、21.23%±0.20%。
基于上述实验结果,28株芽孢杆菌均有产酶、产肽及抗氧化能力,利用因子分析法对28株芽孢杆菌进行综合评价。
为了确定不同芽孢杆菌抗氧化能力指标数据是否适合进行因子分析,首先进行了KMO和Bartlett球形检验和分析,由表6可知,KMO值、Bartlett球形检验分别为0.844、0.0001,变量间相关性较大,数据满足总体正态分布,可以进行因子分析。
用SPSS软件进行因子分析,由表7可知,提取1个公因子时,累计贡献率为97.99%,提取2个公因子时,累计贡献率为99.07%,可综合大部分信息,提取2个公因子即可。
各因子权重如表8所示,得分模型F1=0.462×蛋白酶活力+0.854×超氧化物歧化酶活力+0.025×肽含量−2.227×ABTS+自由基清除率+1.612×DPPH自由基清除率;F2=−0.206×蛋白酶活力−0.639×超氧化物歧化酶活力+0.274×肽含量+2.747×ABTS+自由基清除率−1.474×DPPH自由基清除率。
按各公因子对应的方差贡献率计算F值并进行排序,如表9所示,28株芽孢杆菌中,B. siamensis s4、B. amyloliquefaciens 10、B. subtilis 63、B. subtilis SNBS-3评分较高,分别为1.55、1.53、1.53、1.52,具有高产抗氧化肽潜力;B. amyloliquefaciens 104、B. amyloliquefaciens 25评分较低,分别为−2.01、−2.06,制备抗氧化肽的能力较弱。因此选定B. siamensis s4、B. amyloliquefaciens 10、B. subtilis 63、B. subtilis SNBS-3进行后续超声胁迫。
图1所示为B. subtilis SNBS3、B. amyloliquefaciens 10在UAF方式下(菌株比例1:1、接种量1%、接种4 h后在超声功率120 W/L、超声频率45 kHz)超声10 min后再发酵36 h的蛋白酶活力、肽含量及抗氧化能力变化。
结果表明超声后B. subtilis SNBS-3、B. amyloliquefaciens 10、B. subtilis 63、B. siamensis s4抗氧化能力均有显著提高(P<0.05),其中B. amyloliquefaciens 10的ABTS+自由基清除率及DPPH自由基清除率提高幅度最大,分别为8.69%±0.11%、3.38%±0.81%,但是对于多肽含量和蛋白酶活力并不是具有一定正相关性,不同菌株或菌种对超声的耐受性不同。例如,B. subtilis s4蛋白酶含量增加,但多肽含量下降,表明超声处理可能影响了菌株的基因表达调控网络,激活了与蛋白酶合成相关的基因,使蛋白酶合成增加,活力上升。而蛋白酶在该菌代谢网络中处于关键节点,其活力上升导致多肽作为下游产物被大量消耗,从而含量下降[33]。而在UAF处理后,B. subtilis 63的多肽含量上升,蛋白酶活力下降,其原因可能是超声的机械效应和热效应改变了蛋白酶的高级结构,使其活性位点扭曲或遭到破坏,进而导致酶活力降低。然而,该菌内多肽的生成并非完全依赖于这种蛋白酶的作用。细胞内一些储存多肽的结构因超声作用而被打开,使得原本存在于细胞特定部位的多肽释放出来,从而导致检测到的多肽含量上升,与Ruan等[34]的研究结果一致。综上所述,筛选出超声前后蛋白酶活力、肽含量及抗氧化能力均呈正增长的菌株B. subtilis SNBS-3、B. amyloliquefaciens 10。
本研究以豆渣为发酵基质,筛选出4株具有产抗氧化肽能力的芽孢杆菌。并对UAF过程中的蛋白酶活力、肽含量、抗氧化能力间的关系进行系统研究。最终结果表明,在超声胁迫下B. subtilis SNBS-3和B. amyloliquefaciens 10两株菌的蛋白酶活力、肽含量及抗氧化能力均有显著提高(P<0.05),两株菌具有较强生产抗氧化肽的潜力,本研究对提升豆制品副产物利用率,低成本、高安全、高效率制备活性肽具有重要的意义,可为产业化制备抗氧化肽提供一定的理论支持。但目前对抗氧化肽的研究尚处于起步阶段,多数成果仅停留在实验室阶段,未来针对抗氧化肽的作用机制,仍需深入探究,同时进一步优化生产工艺,提高抗氧化肽生产的效率和稳定性,扩大其应用范围。将有助于将实验室的抗氧化肽研究转化为实际的工业应用,推动食品、化妆品和制药等相关行业的发展。

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doi: 10.13386/j.issn1002-0306.2024120181
  • 接收时间:2024-12-16
  • 首发时间:2026-10-24
  • 出版时间:2026-01-01
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  • 收稿日期:2024-12-16
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    1沈阳农业大学食品学院,辽宁沈阳 110866
    2辽宁省食品发酵技术工程研究中心,辽宁沈阳 110866
    3沈阳市微生物发酵技术创新重点实验室,辽宁沈阳 110866
    4辽宁帝华味精食品有限公司,辽宁葫芦岛 125200

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武俊瑞(1977−)(ORCID:0000−0003−3419−0108),男,博士,教授,研究方向:食品生物技术,E-mail:。
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2种不同金属材料的力学参数
科
Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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