Species-level enumeration of low-concentration lactic acid bacteria in breast milk
It is crucial to identify the distribution of lactic acid bacteria (LAB) in breast milk which is a source of live probiotics for the infant gut. Quantitative PCR (qPCR) is a powerful technique for selectively counting LAB at the species or strain level. However, the limit of quantification (LoQ) of...
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Elsevier
2025-12-01
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| Series: | Food Chemistry: Molecular Sciences |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666566225000309 |
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| author | Yue Sun Qiming Li Mengling Hu You Wu Yunchao Wa Hai Lu Shilong Jiang Hui Wang Qiaosi Wei Yujun Huang Ruixia Gu Chenchen Zhang |
| author_facet | Yue Sun Qiming Li Mengling Hu You Wu Yunchao Wa Hai Lu Shilong Jiang Hui Wang Qiaosi Wei Yujun Huang Ruixia Gu Chenchen Zhang |
| author_sort | Yue Sun |
| collection | DOAJ |
| description | It is crucial to identify the distribution of lactic acid bacteria (LAB) in breast milk which is a source of live probiotics for the infant gut. Quantitative PCR (qPCR) is a powerful technique for selectively counting LAB at the species or strain level. However, the limit of quantification (LoQ) of qPCR is inadequate for human milk samples. To address this issue, nested primer pairs for Lactobacillus and Bifidobacterium species were designed to enable 15 cycles of preamplification Subsequent qPCR assays using the preamplification products (with 10-fold dilution) as templates reduced the LoQ for enumeration plasmids to one-thousandth of the original concentration. Importantly, preamplification did not enhance the detection capability for low biomass samples because the efficiency of DNA extraction was too low. To mitigate the variation in bacteria concentration, inert bacteria were added to the samples. Incorporating Lactococcus lactis at 107 CFU mL−1 into the samples helped expand the linear range between the cycle threshold values and the concentration of target bacteria. By combining preamplification with inert Lc. lactis, probiotic LAB cells could be detected at single-digit levels in breast milk, while also minimizing the requirements for primer quality and DNA extraction kits. This qPCR-based approach is a reliable tool for studying the potential distribution patterns of LAB in low biomass samples, and it enabled approximately a tenfold increase in the number of breast milk samples in which Lactobacillus and Bifidobacterium species were detected, indicating the ubiquitous Lactobacillus and Bifidobacterium in breast milk. |
| format | Article |
| id | doaj-art-4451a70972944d7d83cebb80c7e76079 |
| institution | OA Journals |
| issn | 2666-5662 |
| language | English |
| publishDate | 2025-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Food Chemistry: Molecular Sciences |
| spelling | doaj-art-4451a70972944d7d83cebb80c7e760792025-08-20T02:35:21ZengElsevierFood Chemistry: Molecular Sciences2666-56622025-12-011110026910.1016/j.fochms.2025.100269Species-level enumeration of low-concentration lactic acid bacteria in breast milkYue Sun0Qiming Li1Mengling Hu2You Wu3Yunchao Wa4Hai Lu5Shilong Jiang6Hui Wang7Qiaosi Wei8Yujun Huang9Ruixia Gu10Chenchen Zhang11School of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR ChinaDairy Nutrition and Function Key Laboratory of Sichuan Province, New Hope Dairy Co., Ltd., Chengdu 610023, PR ChinaSchool of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR ChinaSchool of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR ChinaSchool of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR ChinaDivision of Chemical Metrology and Analytical Science, National Institute of Metrology, Beijing 100029, PR ChinaHeilongjiang Feihe Dairy Co., Ltd, Beijing 100016, PR ChinaSchool of Public Health, Shanghai Jiao Tong University, Shanghai 200025, PR China.Heilongjiang Feihe Dairy Co., Ltd, Beijing 100016, PR ChinaSchool of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR ChinaSchool of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR ChinaSchool of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China; Jiangsu Provincial Key Laboratory of Probiotics and Dairy Deep Processing, Yangzhou University, Yangzhou 225127, PR China; Corresponding author at: School of Food Science and Engineering, Yangzhou University, 196 Huayang Xilu, Yangzhou 225100, PR China.It is crucial to identify the distribution of lactic acid bacteria (LAB) in breast milk which is a source of live probiotics for the infant gut. Quantitative PCR (qPCR) is a powerful technique for selectively counting LAB at the species or strain level. However, the limit of quantification (LoQ) of qPCR is inadequate for human milk samples. To address this issue, nested primer pairs for Lactobacillus and Bifidobacterium species were designed to enable 15 cycles of preamplification Subsequent qPCR assays using the preamplification products (with 10-fold dilution) as templates reduced the LoQ for enumeration plasmids to one-thousandth of the original concentration. Importantly, preamplification did not enhance the detection capability for low biomass samples because the efficiency of DNA extraction was too low. To mitigate the variation in bacteria concentration, inert bacteria were added to the samples. Incorporating Lactococcus lactis at 107 CFU mL−1 into the samples helped expand the linear range between the cycle threshold values and the concentration of target bacteria. By combining preamplification with inert Lc. lactis, probiotic LAB cells could be detected at single-digit levels in breast milk, while also minimizing the requirements for primer quality and DNA extraction kits. This qPCR-based approach is a reliable tool for studying the potential distribution patterns of LAB in low biomass samples, and it enabled approximately a tenfold increase in the number of breast milk samples in which Lactobacillus and Bifidobacterium species were detected, indicating the ubiquitous Lactobacillus and Bifidobacterium in breast milk.http://www.sciencedirect.com/science/article/pii/S2666566225000309Quantitative PCRLactic acid bacteriaLow-concentrationPreamplificationInert bacteria |
| spellingShingle | Yue Sun Qiming Li Mengling Hu You Wu Yunchao Wa Hai Lu Shilong Jiang Hui Wang Qiaosi Wei Yujun Huang Ruixia Gu Chenchen Zhang Species-level enumeration of low-concentration lactic acid bacteria in breast milk Food Chemistry: Molecular Sciences Quantitative PCR Lactic acid bacteria Low-concentration Preamplification Inert bacteria |
| title | Species-level enumeration of low-concentration lactic acid bacteria in breast milk |
| title_full | Species-level enumeration of low-concentration lactic acid bacteria in breast milk |
| title_fullStr | Species-level enumeration of low-concentration lactic acid bacteria in breast milk |
| title_full_unstemmed | Species-level enumeration of low-concentration lactic acid bacteria in breast milk |
| title_short | Species-level enumeration of low-concentration lactic acid bacteria in breast milk |
| title_sort | species level enumeration of low concentration lactic acid bacteria in breast milk |
| topic | Quantitative PCR Lactic acid bacteria Low-concentration Preamplification Inert bacteria |
| url | http://www.sciencedirect.com/science/article/pii/S2666566225000309 |
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