具有抑制蓝靛果致腐真菌生长的乳酸菌筛选、鉴定及保鲜研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0107

摘要/Abstract

摘要：

目的筛选并鉴定一株具有抑制蓝靛果采后致腐真菌功能的乳酸菌，并研究其对蓝靛果采后保鲜效果的影响，为解决蓝靛果采后腐烂问题提供新的技术手段。方法从泡菜与发酵浆果中分离纯化乳酸菌后，筛选能抑制蓝靛果采后致腐真菌（链格孢霉与小孢根霉）的乳酸菌P-2，通过16S rRNA基因序列分析鉴定菌株。评估P-2的抗生素敏感性以及不同条件下无细胞上清液（cell-free supernatant, CFS）对致腐真菌抑制效果。最后以P-2的CFS作为保鲜剂对蓝靛果进行保鲜实验。结果在分离并纯化出的22株乳酸菌中，P-2菌株展现出最佳的抑菌效果，经鉴定为乳酸片球菌（Pediococcus acidilactici）。乳酸片球菌P-2对卡那霉素、庆大霉素与链霉素耐受，对其他抗生素敏感或中性敏感，其CFS在酸性条件下的抑菌活性较强，对蛋白酶敏感。P-2 CFS对链格孢霉和小孢根霉的MIC分别为12.8 mg/mL、25.6 mg/mL。与对照组相比，乳酸片球菌P-2的CFS可以降低蓝靛果的失重率和腐烂率，减缓蓝靛果在贮藏期间硬度、总酚、类黄酮以及维生素C含量的下降，同时可以使蓝靛果的货架期至少延长7 d。结论乳酸片球菌P-2的CFS具有抑制蓝靛果致腐真菌的作用，可作为一种绿色保鲜剂应用于浆果的采后保鲜中。

关键词: 乳酸菌, 蓝靛果, 致腐真菌, 抗真菌活性, 链格孢霉菌, 小孢根霉菌, 筛选, 保鲜

Abstract:

Objective To screen and identify a strain of lactic acid bacteria that can inhibit rot-causing fungi in post-harvest Lonicera caerulea L., and to explore its effect on post-harvest preservation of L. caerulea L, this may provide new technical means to solve the problem of post-harvest rotting of L. caerulea L. Method After the isolation and purification of lactic acid bacteria from pickles and fermented berries, the P-2, which can inhibit the rot-causing fungi (Alternaria alternata and Rhizopus microsporus) in post-harvest L. caerulea L., were screened and identified by 16S rRNA gene sequence analysis. To evaluate the antibiotic sensitivity of P-2 and the inhibitory effect of cell-free supernatant (CFS) on rot-causing bacteria under different conditions. At last, P-2 CFS was used as a preservative to keep L. caerulea L fresh. Result Among the 22 strains of isolated and purified lactic acid bacteria, P-2 strain showed the best bacteriostatic effect and was identified as Pediococcus acidilactici. P-2 is resistant to kanamycin, gentamicin and streptomycin, sensitive or neutral to other antibiotics, its CFS has strong antifungal activity under acidic conditions, and is sensitive to protease. The MIC of P-2 CFS against A. alternata and R. microsporus were 12.8 and 25.6 mg/mL, respectively. Compared with the control group, the CFS of P-2 reduced the weight loss rate and decay rate of L. caerulea L, slowed down the decline of hardness, total phenol, flavonoid and vitamin C content of L. caerulea L during storage, and prolonged the shelf life of L. caerulea L. for at least 7 d. Conclusion The CFS of P. acidilactici P-2 can inhibit the post-harvest rot-causing bacteria of L. caerulea L., and can be used as a green preservative in the post-harvest preservation of berries.

Key words: lactic acid bacteria, Lonicera caerulea L., rot-causing fungi, antifungal activity, Alternaria alternata, Rhizopus microspora, screening, keep fresh

李德海, 殷丽, 周才雪, 王泽童, 孙常雁. 具有抑制蓝靛果致腐真菌生长的乳酸菌筛选、鉴定及保鲜研究[J]. 生物技术通报, 2025, 41(8): 289-299.

LI De-hai, YIN Li, ZHOU Cai-xue, WANG Ze-tong, SUN Chang-yan. Screening， Identification and Preservation of Lactic Acid Bacteria Inhibiting the Growth of Rot-causing Fungus Lonicera caerulea L.[J]. Biotechnology Bulletin, 2025, 41(8): 289-299.

图/表 10

表1 乳酸菌对链格孢霉和小孢根霉的抗真菌活性

Table 1 Antifungal activities of lactic acid bacteria against Alternaria alternata and Rhizoctonia microspora

乳酸菌 Lactic acid bacteria	链格孢霉 A. alternata	小孢根霉 R. microspora
P-1	-	-
P-2	+++	++
P-3	-	-
P-4	++	++
P-5	++	+++
P-6	+++	-
P-7	-	+
P-8	-	-
P-9	++	+
P-10	+	-
P-11	+	-
J-12	+++	++
J-13	++	+++
J-14	-	-
J-15	-	++
J-16	++	-
J-17	+	-
J-18	-	-
J-19	++	++
J-20	+	-
J-21	++	++
J-22	-	-

图1 不同乳酸菌CFS对小孢根霉（A）与链格孢霉（B）的抗真菌活性筛选

Fig. 1 Screening of antifungal activities of cell-free supernatants from different lactic acid bacteria against R. microspora (A) and A. alternata (B)

图2 菌株P-2的16S rRNA PCR扩增产物琼脂糖凝胶电泳图（A）和系统发育树（B）

Fig. 2 Agarose gel electrophoresis of 16S rRNA PCR products of strain P-2 (A) and phylogenetic tree (B)

表2 乳酸片球菌P-2抗生素敏感性判断标准与结果

Table 2 Judgment criteria and results of antibiotic sensitivity of Pediococcus acidilactici P-2

抗生素 Antibiotic	抑菌直径 Inhibitory diameter （mm）			P-2抑菌直径 P-2 inhibitory diameter （mm）	结果 Result
抗生素 Antibiotic	敏感 Susceptible （S）	中介 Intermediary （I）	耐受 Resistance （R）	P-2抑菌直径 P-2 inhibitory diameter （mm）	结果 Result
四环素 Tetracycline	≥19	15-18	≤14	24.43±1.39^e	S
氯霉素 Chloramphenicol	≥18	13-17	≤12	40.83±0.67^b	S
氨苄西林 Ampicillin	≥17	14-16	≤13	32.97±0.73^c	S
万古霉素 Vancomycin	≥17	15-16	≤14	15.50±0.12^f	I
庆大霉素 Gentamicin	≥15	13-14	≤12	0.00±0.00^g	R
卡那霉素 Kanamycin	≥18	14-17	≤13	0.00±0.00^g	R
链霉素 Streptomycin	≥15	12-14	≤11	0.00±0.00^g	R
红霉素 Erythromycin	≥23	14-22	≤13	29.50±0.70^d	S
克林霉素 Clindamycin	≥21	15-20	≤14	42.97±0.58^a	S

图3 不同温度处理乳酸片球菌P-2 CFS对小孢根霉（A）、链格孢霉（B）的抑制效果

Fig. 3 Inhibition effects of CFS of Pediococcus acidilactici P-2 treated at different temperatures on R. microsporus (A) and A. alternata (B)

图4 不同酶处理对乳酸片球菌P-2 CFS对小孢根霉（A）、链格孢霉（B）的抑制效果

Fig. 4 Inhibitory effect of CFS of Pediococcus acidilactici P-2 treated with different enzymes on R. microsporus (A) and A. alternata (B)

图5 不同pH条件下乳酸片球菌P-2 CFS对小孢根霉（A）、链格孢霉（B）的抑制效果

Fig. 5 Inhibition effect of CFS of P. acidilactici P-2 under different pH conditions on R. microsporus (A) and A. alternata (B)

图6 乳酸片球菌P-2 CFS对小孢根霉（A）和链格孢霉（B）最小抑菌浓度（MIC）

Fig. 6 Minimum inhibitory concentration (MIC) of CFS from P. acidilactici P-2 against R. microsporus (A) and A. alternata (B)

图7 不同浓度乳酸菌P-2 CFS处理对蓝靛果失重率、腐败率、硬度、总酚含量、类黄酮含量与VC含量的影响不同字母表示不同处理差异显著（P<0.05）。下同

Fig. 7 Influences of different concentrations of Pediococcus acidilactici P-2 CFS on the weight loss rate, spoilage rate, hardness, total phenolic content, flavonoid content and vitamin C content of Lonicera caerulea L.Different letters indicate significant differences in different treatment methods (P<0.05). The same below

表3 对蓝靛果保鲜过程中的感官评价

Table 3 Sensory evaluation of L. caerulea L. during storage

处理组 Processing group	时间 Time （d）	外观 Appearance （Scores）	质地 Texture （Scores）	味道 Taste （Scores）	整体可接受性 Overall acceptability （Scores）
CK	1	4.20±0.00^a	4.80±0.45^a	4.60±0.55^a	5.00±0.00^a
	7	4.00±0.00^a	1.80±0.84^b	1.60±0.55^b	2.20±0.45^b
	14	-	1.20±0.45^b	-	-
	21	-	0.20±0.45^c	-	-
	28	-	-	-	-
1/2 MIC	1	4.60±0.55^a	5.00±0.00^a	4.80±0.45^a	4.60±0.55^a
	7	4.20±0.84^a	4.00±0.71^b	3.20±1.64^a	4.40±0.89^a
	14	3.20±1.30^ab	2.40±0.55^c	1.20±0.84^b	2.80±0.84^b
	21	1.60±1.14^bc	0.80±0.45^d	0.20±0.45^b	1.20±0.84^c
	28	0.2±0.45^c	0.20±0.45^d	-	0.40±0.89^c
MIC	1	4.60±0.55^a	4.80±0.45^a	4.60±0.55^a	4.80±0.45^a
	7	4.20±0.45^ab	3.60±0.55^a	3.20±0.45^b	3.80±0.45^a
	14	3.40±0.55^b	2.00±1.00^b	1.20±0.84^c	2.40±0.55^b
	21	2.20±0.84^c	1.40±1.14^b	0.40±0.55^cd	1.00±1.00^c
	28	0.40±0.55^d	0.60±0.55^b	-	0.20±0.45^c
2 MIC	1	4.60±0.55^a	4.8±0.45^a	4.60±0.55^a	5.00±0^a
	7	4.40±0.55^a	4.00±0.71^a	3.80±0.45^a	4.20±0.45^a
	14	3.60±0.55^a	2.00±1.00^b	1.80±0.84^b	2.80±0.45^b
	21	2.20±0.84^b	1.40±1.14^b	1.00±0.71^bc	1.40±0.89^c
	28	0.60±0.5^c	0.80±0.45^b	-	1.00±0.71^c

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