The upward trends of vaccine exemptions in Texas are alarming. While HPV vaccine rates in this State are among the lowest nationwide, factors that contribute to the low HPV vaccination uptake among adults remain unknown. In this study, we examined the main reasons for not receiving HPV vaccination among age‐eligible adults.The Texas health screening survey (2018), a multistage area probability design‐based survey of a representative sample of Texas residents, was used to identify 907 eligible adults (age ≥ 18 years) respondents, including 724 women aged ≤ 26 years in 2007 (≤38 years in 2018), and 183 men aged ≤ 21 years in 2011 (≤28 years in 2018). Participants who reported having never received an HPV shot, where asked the main reason for not receiving the vaccine.Overall, 58.5% (95%CI: 55.1‐62.0) of vaccine eligible adults reported having never received the HPV vaccine. The most commonly reported reasons for not receiving it were: did not know about the vaccine (18.5% (14.9‐22.1)), and provider did not recommend (14.1% (10.9‐17.4)). In contrast, commonly perceived reasons such as: safety concerns (7.2% (4.8‐9.5)), lack of insurance (3.4% (1.7‐5.1), and concerns about increasing sexual activity if vaccinated (0.2% (0.0‐0.5)), were less frequently reported.Among vaccine‐eligible adults, safety and sexuality concerns do not appear to be the prime factors underlying low HPV vaccination rates. Rather than emphasizing them, educational interventions should aim at improving vaccine's knowledge, and enhancing provider recommendations on the necessity of HPV vaccination. Among HPV‐vaccine eligible adults, lack of knowledge about the vaccine and lack of provider recommendations are the most commonly reported reasons for not receiving the HPV vaccine. In this population, educational interventions should focus on enhancing HPV vaccine's knowledge and provider recommendations of the necessity and awareness of HPV vaccination. Vaccines to prevent some HPV infections are available for men and women 9 ‐ 26 years of age and are sometimes called the HPV shot, Cervarix, or Gardasil. Have you ever received the HPV shot, Cervarix, or Gardasil? The possible responses were: Yes/No/Don't know. Participants who responded “No” were asked: (b) What is the main reason you have not received the HPV shot/ vaccine? Possible responses were: Provider did not recommend (a), Did not know about the vaccine (b), Vaccine not needed or necessary (c), Not required to get the vaccine (d), Safety concerns (e), Provider indicated could vaccinate later (f), Uninsured or insurance doesn't fully cover shots, or co‐pay or other costs too high (g), Shot could be painful (h), Vaccine not available (i), Difficulty making or getting to appointments/ transportation issues (j), Concern about increasing sexual activity if receive shot (k), Not sexually active (l), Some other reason (m).The main outcomes for this study were: (a) self‐reported receipt of HPV vaccine, and (b) self‐reported reasons for not receiving HPV vaccine. To measure these outcomes, we adapted two validated questions from the National Immunization Survey‐Teen (NIS‐Teen). In the Texas health screening survey, these questions were directed to adult respondents who were or have been eligible to receive HPV vaccination. We first asked: (a) 2.3Covariates were selected based on their potential influence on HPV vaccination uptake, including: socio‐demographic factors of age, ethnicity/race, nativity, educational attainment, marital status, occupation, rurality, and household income; behavioral and mental factors; and health‐related variables such as smoking status, health coverage, vaccination against Hepatitis B virus (HBV), hormonal contraception use, and family history of any cancer.2.4Data were weighted by ICF International, Inc, Fairfax, Virginia using a three‐dimensional raking approach with iterative post‐stratification based on sex, age, and race/ethnicity.33.1Table HPV unvaccinated vs vaccinated respondents were more frequently: aged between 29 and 38 years [36.2% 31.9‐40.5) vs 21.1% (15.3‐26.9)]; males [31.5% (26.7‐36.2) vs [26.7% (19.1‐34.4)], and NHWs [31.5% (27.0‐36.1) vs 27.0% (19.7‐34.3)]. HPV unvaccinated respondents were also more likely to: have a level of education not greater than 12 years [35.1% (30.6‐39.5) vs 27.7% (20.9‐34.4)]; and to reside in rural areas [(39.8% (35.2‐44.4) vs 34.9% (27.5‐42.4)], compared with their vaccinated counterparts.  vs 57.1% (49.4‐64.8)]; have health insurance [62.7% (58.2‐67.2) vs 68.5% (61.3‐75.6)]; be vaccinated against HBV [33.1% (28.7‐37.4) vs 73.7% (67.1‐80.4)]; and have a family history of cancer [50.8% (46.1‐55.4) vs 66.9% (59.7‐74.0)], compared with HPV vaccinated respondents. ); “uninsured or insurance doesn't fully cover shots” (3.4% 1.7‐5.1)); “concerns about increasing sexual activity if receive the vaccine” [0.2% (0.0‐0.5)]; and “difficulty making or getting to appointments, or transportation issues” (0.2% (0.0‐0.4)) ; Reasons for not having received HPV vaccination described according to respondents’ characteristics age, origin of birth, marital status, occupational status, income, residency, use of hormonal contraception, smoking status, availability of health care, vaccination status for hepatitis B, and family history of cancer are reported in the Table 4In this representative sample of adult Texas residents eligible for HPV vaccination, differences in socio‐demographic, health‐related, and behavioral characteristics were assessed between those who reported having ever received the HPV vaccine, and those who had never been vaccinated for HPV. Among eligible respondents who reported having not received HPV vaccination, we examined the reasons why they did not get the vaccine. To the best of our knowledge, this is the first study to assess individual's reasons for not receiving HPV vaccine in the Texas adult population, a state with an alarmingly low HPV vaccine uptake.Texas has experienced a 20‐fold increase in nonmedical exemptions of vaccination for students since 2003 when exemptions for religious and personal beliefs were enacted.HPV vaccination is routinely administered between 11 and 12 years with parental consent (2). However, catch‐up immunization is recommended for both men and women through early adulthood,In our study sample, lack of knowledge about HPV vaccine, lack of provider's recommendation, and perceived lack of necessity, were the most commonly reported reasons for not getting the vaccine. Interestingly, these reasons varied by certain demographics. Among women, the main reason for not getting the HPV vaccine was “did not know about the vaccine”, while among men, it was “not sexually active.” In a study assessing US parents’ reasons for lack of HPV vaccine initiation in their children, “safety concerns” was most frequently reported by parents of eligible girls vs “lack of necessity” by parents of eligible boys.Although the HPV vaccine was FDA approved to prevent cancer over a decade ago, eligible adults still report a lack of knowledge and understanding of its necessity. However, safety concerns, lack of insurance, transportation issues as well as some other concerns were less common in our study sample, suggesting that reasons commonly highlighted in mainstream media, such as anti‐vaccination beliefs including religious objections are not the prime factors underlying individuals’ perception of HPV vaccination in Texas.5Strengths of the study include its large sample size and the representativeness of the adult Texas population. The study is somewhat limited, however, in its ability to detect simultaneous reasons individuals may hold, as the question was framed to assess the “main” reason for not getting the vaccine. Because information about whether or how often eligible individuals visited a healthcare provider was not available, we could not distinguish between respondents with missed clinical opportunities for HPV vaccine recommendation, and those unexposed to provider recommendations. While the self‐report of the vaccine status by study participants could yield a certain level of “misclassification” bias, substantial agreement between electronic medical reports of the vaccine status and self‐reported data were noted for HPV vaccination.6In this study, individual reasons for not receiving the HPV vaccine were assessed among eligible adults. A substantial proportion of age‐eligible adults in Texas reported lack of knowledge or lack of provider recommendation as the main reasons for not receiving HPV vaccine. Rather than emphasizing on safety or sexual activity, educational interventions should focus on the necessity and awareness of HPV vaccination. Providers’ recommendation for HPV vaccine should be reinforced, and further adapted to older age groups.The authors declare no potential conflicts of interest.Concept and Study Design: Fokom Domgue, Shete. Data Analysis: Yu. Interpretation of the data: Fokom Domgue, Cunningham, Yu, Shete. Initial Draft: Fokom Domgue. Critical revision of the manuscript for important intellectual content: Fokom Domgue, Cunningham, Yu, Shete. Obtained Funding and Study Supervision: Shete.The funders had no involvement in design and conduct of the study; analysis and interpretation of the data; preparation, review, or approval of the manuscript; and the decision to submit the manuscript for publication.TableS1Click here for additional data file.


Preoperatively and 6 months postoperatively, we compared the corneal endothelial cell density (ECD), the coefficient of variation in cell size (CV), and the percentage of hexagonal cells (HEX), in the peripheral regions of the cornea, using a non-contact specular microscope .p = 0.16, nasal superior, p = 0.12, temporal superior, p = 0.17, inferior, p = 0.37, nasal inferior, p = 0.28, temporal inferior, p = 0.17). The mean percentage of the ECD loss was 1.3, 1.3, 1.0, 1.4, 0.7, and 1.4%, respectively. No significant differences in the peripheral CV or HEX were found preoperatively and 6 months postoperatively at each point.All keratoconic eyes in this series were measurable in the peripheral regions. No significant differences were found in the peripheral ECD preoperatively and 6 months postoperatively at each point .University Hospital Medical Information Network Clinical Trial Registry ( Corneal cross-linking (CXL) has been acknowledged as an effective method for halting the progression of keratoconus by photosensitization of riboflavin activated by ultraviolet-A (UV-A) –6. For t2 difference in the ECD, when the standard deviation (SD) of the mean difference was 160 cells/mm2. All patients received standard CXL treatment for progressive keratoconus at Kitasato university hospital, and completed a 6-month follow-up. The patients were recruited in a continuous cohort. Diagnosis of keratoconus was performed by one experienced clinician (K.K.) with evident findings characteristic of keratoconus , and at least one keratoconus sign  on slit-lamp examination [We registered the study protocol with the University Hospital Medical Information Network Clinical Trial Registry 000031162). We enrolled thirty-one eyes of 31 patients (12 men and 19 women) in this retrospective study. The sample size in this study offered 92.0% statistical power at the 5% level in order to detect a 100-cells/mm1162. We 2 by the instrument’s built-in software for cell analysis.Preoperatively and 6 months postoperatively, we quantitatively evaluated the ECD, the coefficient of variation in cell size (CV), and the percentage of hexagonal cells (HEX), in the peripheral regions  of the cornea, using a non-contact specular microscope . Patients were asked to fixate at each peripheral target, and photographs were taken of the peripheral corneal endothelium using the auto-alignment function. The system automatically measures the area of corneal endothelial cells (> 100 cells), and after that it automatically calculates the ECD, CV, and HEX within an area of 0.1 mm2 was performed for 30 min with an OptoXLink corneal cross-linking system™ . Riboflavin solution was administered every 5 min during the irradiation. We used topical steroidal and antibiotic medications 4 times daily for 2 weeks after treatment, with the dose being reduced gradually thereafter, with a soft contact lens until re-epithelialization.The standard CXL technique was performed in accordance with the Dresden protocol . Topicalp < 0.05 was considered statistically significant, unless otherwise indicated.All statistical analyses were performed with a commercially available statistical software . In order to compare the pre- and the post-surgical data in the peripheral regions, we performed the Wilcoxon signed-rank test. The results are shown as mean ± SD, and a value of The preoperative patient demographics are summarized in Table p = 0.16, nasal superior, p = 0.12, temporal superior, p = 0.17, inferior, p = 0.37, nasal inferior, p = 0.28, temporal inferior, p = 0.17). The mean percentage of the ECD loss was 1.3, 1.3, 1.0, 1.4, 0.7, and 1.4%, respectively.The preoperative and postoperative ECD in the peripheral regions are shown in Fig. p = 0.23, nasal superior, p = 0.21, temporal superior, p = 0.11, inferior, p = 0.15, nasal inferior, p = 0.21, temporal inferior, p = 0.19).The preoperative and postoperative CV in the peripheral regions are shown in Fig. p = 0.14, nasal superior, p = 0.80, temporal superior, p = 0.30, inferior, p = 0.49, nasal inferior, p = 0.38, temporal inferior, p = 0.64).The preoperative and postoperative HEX in the peripheral regions are shown in Fig. The results of this study showed that the mean peripheral ECD loss was 0.7 to 1.4% 6 months after CXL, and that there were no significant changes in the peripheral ECD before and after CXL. This indicates that the standard CXL treatment was safe in terms of the peripheral ECD. This study also demonstrated that there were no significant changes in corneal morphological parameters such as CV or HEX, indicating that the CXL treatment did not cause a significant change in peripheral polymegethism or polymorphism. To the best of our knowledge, this is the first study on the detailed analysis of the peripheral endothelial cell density and morphology before and after CXL treatment.Table In this study, we found no significant changes in the peripheral CV or HEX after CXL, which was the same as previous studies with no significant changes in the central CV or HEX –19, 21. There are several research limitations that affect this study. First, the study was conducted in a retrospective fashion. Second, the sample size was relatively small. Third, the follow-up period is up to 6 months. A further long-term prospective study with a large cohort of patients undergoing CXL is necessary to confirm our preliminary findings.In summary, our results support the view that the peripheral ECD, CV and HEX remained stable during the 6-month follow-up in post-CXL eyes. Based on the findings of this study, it is suggested that CXL did not cause a significant change in the density or the morphology of the peripheral corneal endothelial cells, and it is therefore a minimally invasive surgical approach, in terms of peripheral endothelial cells, for progressive keratoconus.


Aspergillus oryzae β-galactosidase is reported for the first time. The effect of operational variables: inlet concentrations of sugar substrates, temperature, feed substrate molar ratio, enzyme loading and feed flow rate was studied on reactor performance. Even though the variation of each one affected to a certain extent lactulose yield (YLactulose), specific productivity (πLactulose) and selectivity of the reaction  (SLu/TOS), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum YLactulose of 0.54 g⋅g–1 was obtained at 50°C, pH 4.5, 50% w/w inlet concentrations of sugar substrates, feed flowrate of 12 mL⋅min–1, fructose/lactose molar ratio of 8 and reactor enzyme load of 29.06 IUH⋅mL–1. At such conditions SLu/TOS was 3.7, lactose conversion (XLactose) was 0.39 and total transgalactosylation yield was 0.762 g⋅g–1, meaning that 76% of the reacted lactose corresponded to transgalactosylation and 24% to hydrolysis, which is a definite advantage of this mode of operation. Even though XLactose in CSTR was lower than in other reported modes of operation for lactulose synthesis, transgalactosylation was more favored over hydrolysis which reduced the inhibitory effect of galactose on β-galactosidase.Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized  Aspergillus oryzae produced the highest yield, productivity and selectivity of lactulose synthesis.Lactulose  is a synthetic disaccharide mainly used in the pharmaceutical field as a drug for the treatment of chronic constipation and hepatic encephalopathy . IndustrSeveral types of reactors and modes of operation have been studied  with the purpose of increasing yield, productivity and selectivity of lactulose synthesis, using both soluble and immobilized β-G . The higEven though batch synthesis is easy to implement, it has the disadvantage of using a high amount of enzyme and the control of the reaction becomes problematic, since reaction kinetics should be precisely monitored (or modeled) in order to stop the reaction at the moment of maximum lactulose yield, before hydrolysis takes over . BesidesH⋅g–1) and operational stability, reporting a half-life of 2823 that corresponds to a stabilization factor  of 29.4 at fructose-lactose molar ratio of 4 , o-nitrophenyl-β-D-galactopyranoside (o-NPG) and galacto-oligosaccharides standards  were supplied by Sigma . Lactulose was provided by Discovery Fine Chemicals . Agarose Bead Standard (6% cross-linked with epichlorohydrin) and packed bed reactor were purchased from Agarose Bead Technologies . The enzyme used was EnzecoTM Fungal Lactase Concentrate, a commercial preparation of A. oryzae β-galactosidase kindly donated by Enzyme Development Corporation, New York, NY, United States. All other reagents were analytical grade and supplied by Sigma or Merck .D (+) Lactose monohydrate, D (+) glucose, D (+) fructose, D (+) galactose, A. oryzae β-G catalysts was determined according to The enzymatic activity of the immobilized A Jasco RI 2031 HPLC delivery system, provided with refractive index detector was used for the quantification of substrates (fructose and lactose) and products of the synthesis of lactulose  according to the protocol reported by A. oryzae β-G in monofunctional glyoxyl-agarose supports was done following the procedure described by H) was defined as the amount of β-G that hydrolyzes 1 μmol of o-NPG per minute at 45 mM o-NPG, 40 °C and pH of 4.5 (H⋅g –1 as defined above.Immobilization of H of 4.5 . The glyE) of 2 L, and the working volume for lactulose synthesis was 585 mL; temperature was kept at 50°C and pH was kept at 4.5. Sugar substrates were dissolved in 100 mM citrate-phosphate buffer pH 4.5 previously heated at 95°C and then cooled down to the reaction temperature. Different fructose/lactose molar ratios (F/L) were fed to the reactor using a Masterflex L/S 7525 (United States) pump and Masterflex 96400-14 silicone tubing connectors cured in peroxide, and 0.5 mL samples at the reactor outlet were taken every hour. Product distribution was determined by analyzing the amounts of lactulose, disaccharides, trisaccharides and tetrasaccharides produced. The assays were carried out in duplicate, with standard deviations always below 5%. Quantification of carbohydrates was carried out as described in See section “Determination of Carbohydrates.”Lactulose), TOS yield (YTOS), total yield of transgalactosylation (YT), lactulose total yield (YLu∗), specific productivity of lactulose synthesis (πLactulose), lactose conversion (XLactose) and selectivity of lactulose synthesis (SLu/TOS) as previously described by The effect of operation variables  were evaluated in the synthesis of lactulose in CSTR operation in terms of the lactulose yield  on YLactulose, YTOS, YT, XLactose, πLactulose, and SLu/TOS in the synthesis of lactulose in CSTR was evaluated at pH 4.5, 50% (w/w) inlet total carbohydrates, enzyme loading in the reactor of 29.06 IUH⋅mL–1, flowrate of 6 mL⋅min–1 and feed F/L of 16. CSTR was operated for 120 h at the different temperatures for determining the operational thermal stability of the biocatalyst. Inactivation profiles of the biocatalysts under operating conditions were modeled according to The effect of temperature  on YLactulose, YTOS, YT, XLactose, πLactulose, and SLu/TOS in the synthesis of lactulose in CSTR was evaluated at 50°C, pH 4.5, 50% (w/w) inlet total carbohydrates, enzyme loading in the reactor of 29.06 IUHmL–1, flowrate of 6, 9, and 12 mL⋅min–1 and F/L of 4, 8, 16, and 24.The effect of F/L  on YH⋅mL–1) on YLactulose, YTOS, YT, XLactose, πLactulose, and SLu/TOS in the synthesis of lactulose in CSTR was evaluated at 50°C, pH 4.5, 50% (w/w) inlet total carbohydrates, flowrate of 6 mL⋅min–1 and F/L of 4, 8, and 16. Enzyme loadings of 17.4, 29.06, and 58.12 IUH⋅mL–1 correspond to catalyst mass to flowrate ratios (mcat⋅F–1) of 0.5, 0.8, and 1.6 g⋅min⋅mL–1, respectively.The effect of enzyme loading in the reactor  in the synthesis of lactulose in CSTR was evaluated at 50°C, pH 4.5, 50% (w/w) inlet total carbohydrates, F/L  and enzyme loading in the reactor of 29.06 IUH⋅mL–1. Flowrates of 3, 6, 9, and 12 mL⋅min–1 correspond to catalyst mass to flowrate ratios (mcat⋅F–1) of 1.6, 0.8, 0.5, and 0.4 g⋅min⋅mL–1, respectively.The effect of flowrate   .T increased significantly with the increase in the inlet sugar substrates concentrations  down to 0.52 at 60% (w/w) (T (X0% (w/w) . These r(w/w)  (T  and for (w/w)  (0% (w/w) .Lu/TOS increased with the increase in the inlet concentrations of sugar substrates (Lactulose and YTOS).Sbstrates , which rLactulose with those reported for other enzymes and modes of operation, lactulose total yield (YLu∗) was defined , being lower than the value of 0.31 g⋅g–1 reported for the soluble enzyme at a F/L of 12, and the value of 0.28 g⋅g–1 obtained with glyoxyl-agarose immobilized A. oryzae β-G under the same operational conditions in batch and in repeated batch operation  of the enzyme and with the enzyme covalently immobilized to the heterofunctional support amino-glyoxyl-agarose , favor transgalactosylation over hydrolysis, and reaction rates are high; however high temperatures will promote subunit dissociation  and in gactulose  similar  to 60°C . The res to 60°C .Lactose, πLu, and SLu/TOS increased slightly with the increase in temperature in the range between 50 and 60°C. Increase in πLu was also reported for the soluble enzyme, temperature increase producing an increase in the reaction rate of lactulose synthesis, therefore reducing reaction time  and (e) are the initial and residual enzyme activity after time (t) respectively, the first-order inactivation rate constant (kD), the specific activity ratio of the final and initial enzyme species (α).where  obtained are presented in 1/2 values are much higher than previously reported for the kinetics of enzyme inactivation under non-reactive conditions.Based on the resulting model, kA. oryzae was well described by a one-stage first-order mechanism of inactivation without residual activity, with t1/2 values of 0.43 h at 60°C. A. oryzae and Bacillus circulans under non-reactive conditions for the immobilization of glyoxyl agarose, reporting that the A. oryzae β-G was much more stable than the B. circulansβ-G with a 3.6 times higher t1/2 at 60°C.A. oryzae β-G under non-reactive conditions obtaining a residual activity of 60% after 40 h at 50°C; however, the glyoxyl-agarose immobilized enzyme at the same conditions retained more than 80% of the initial activity.A. oryzae β-G during repeated batch operation at 50°C, obtaining a t1/2 of 2823 h, while under non-reactive conditions t1/2 was only 96 h, so that a stabilization factor of 29.4 was attained when operating at F/L of 4; however, at F/L of 20 t1/2 was 940 h, being the stabilization factor only 9.7, much lower than at a ratio of 4. Similar results were reported for CLEAs of A. oryzae β-G, where a t1/2 of 1070 h was obtained under reactive conditions and only 123 h at non-reactive conditions, obtaining a stabilization factor of 8.7 at F/L of 4; at F/L of 20 a t1/2 315 h was obtained under reactive conditions, corresponding to a stabilization factor of 2.51, which is quite lower than obtained at lower ratios , a slight increase in yield is observed with the increase in reactor enzyme load at any F/L  was determined, which decreased with the increase in feed flowrate, as shown in catalyst  and thosctis β-G . ConsideLu∗ with the increase in feed flowrate, was also reported by A. oryzae β-galactosidase. They explained that due to the short reaction time, mass transfer of the substrate into the immobilized enzyme molecules was reduced affecting both its transgalactosylation and hydrolysis. The reduction in XLactose  at the three F/L tested. Maximum πLactulose of 0.31 gLactulose⋅ was obtained at 12 mL⋅min–1 and F/L 4. These results agree with those reported by Lactulose increased at shorter HRT due to the higher amount of effluent produced.Lu/TOS decreased with the increase in feed flowrate, this effect being more pronounced at F/L 16 . It is worth mentioning that comparison is not completely fair, since even though the same operation conditions were used, flowrates and sizes of the continuously operated reactors were different. However, Lactulose and YTOS; however, XLactose was the lowest. This means that CSTR allows reducing the hydrolysis of lactose, which is reflected in the higher YLactulose and YTOS obtained, and also reduced the incidence of galactose competitive inhibition on β-G. In CPBR and BSTR, even though XLactose was high, YLactulose and YTOS were low, which reflects the higher incidence of lactose hydrolysis, which is more severe at low feed flowrates  inlet concentration of sugar substrates, feed flowrate 12 mL⋅min–1 and fructose/lactose molar ratios of 8, these values being higher than reported for the synthesis of lactulose in other modes of operation. Results reported in this work represent a first approximation for scaling up the enzymatic production of lactulose as an environmentally compliant alternative to its production by chemical synthesis.Lactulose synthesis in CSTR with glyoxyl-agarose immobilized The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CU, NR, and FV: conceptualization, investigation, and visualization. CV: conceptualization, methodology, writing – original draft preparation, and reviewing and editing. AI: conceptualization, methodology, writing – original draft preparation, and reviewing and editing. CG: conceptualization, methodology, formal analysis, resources, supervision, visualization, original draft preparation, and writing – reviewing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lactobacillus plantarum (strain 178). First, acidification in a meat model medium and bioactivity towards Staphylococcus aureus, Salmonella sp., Listeria monocytogenes, and Escherichia coli were assessed; the performances of Lb. plantarum 178 were compared to those of a commercial Lb. sakei and a probiotic Lb. casei. Lb. plantarum 178 inhibited the pathogens and experienced a higher acidification at 15 °C. Lb. casei and Lb. plantarum were used for an industrial fermentation of traditional Italian sausages. The strains assured the correct course of fermentation and inhibited pathogens and enterobacteria. This study represents the scaling up and the validation of a promising strain at industrial level and shows the possibility of performing the fermentation of traditional Italian sausage through functional starter cultures, combining the benefit of a controlled fermentation and possible health benefits.This paper proposes the industrial validation of a functional strain of  The awareness of consumers of the importance of some foods in diet is increasing, along with the knowledge of the benefits derived from the use of certain microorganisms—therefore, thanks to new biotechnologies, some traditional processes have been modified to improve the quality of the final product. In particular, fermentation processes can be significantly improved by modern technology and biotechnology . FermentEscherichia coli, Listeria monocytogenes, and Yersinia enterocolitica) and/or spoilage microorganisms—in fact, a rapid acidification is important for safety, while a high competitiveness against the spontaneous lactic flora is important for product quality [The fermentation of sausages involves the participation of mainly lactic acid bacteria (LAB), coagulase-negative staphylococci (CNS), yeasts, and molds. LAB converts fermentable sugars to lactic acid by creating unfavorable conditions for pathogens , and functional traits .A solution to overcome this challenge is the use of autochthonous LAB, because they are desirable for: (i) their ability to adapt to the ecological conditions of specific meat fermentations, (ii) control of the ripening processes, and (iii) the ability to inhibit the growth of spontaneous microorganisms—therefore autochthonous starters are recommended to achieve the desired fermentation parameters specific for the product type . In a prLb. plantarum 178 to improve the quality of Italian sausages. The performances of this strain were preliminarily compared to a commercial probiotic (Lb. casei LC01) and to a commercial starter (Lb. sakei ST2), and then validated in an industrial process.The aim of this paper is to propose an industrial validation of Lb. plantarum 178, isolated from pork meat, a functional starter with probiotic characteristics [Lb. casei LC01, Chr. Hansen ; and (iii) Lb. sakei ST2, isolated from a commercial preparation [−1) of sterile glycerol . Before each assay, the microorganisms grown in MRS broth were incubated at 30 °C for 24–48 h.Three strains were used in this research: (i) eristics ; (ii) Lb−1; bacteriological peptone, 10 g·L−1; tryptone, 5 g·L−1; NaNO2, 150 mg·L−1; NaNO3, 250 mg·L−1; NaCl, 50 g·L−1; lactose, 4 g·L−1; black pepper, 1 g·L−1), inoculated to 7 log cfu·mL−1 and incubated at 15, 20, 25, 30 and 44 °C. The pH was measured after 6 and 24 h through a pH meter Crison 2001 .Acidification  was assessed in a meat model system  incubated at 37 °C for 24 h.The interaction of −1 on nutrient agar plates, then wells of 9 mm of diameter were cut off through a cork borer and 80 µL of the following solution was distributed: (i) Solution A: LAB culture in MRS broth (ca. 7 log cfu·mL−1); (ii) Solution B: not-buffered LAB supernatant (cell-free) prepared by centrifuging cell culture at 1000× g for 10 min; and (iii) Solution C: cell-free LAB supernatant buffered to pH 6.0 through NaOH 1 mol L−1. Distilled water was used as a negative control.Pathogens were separately inoculated to 7 log cfu·mLThe tests were performed on three independent batches. The plates were incubated at 15, 25 and 30 °C for 24–48 h. An inhibition zone around the well was assumed to be a significant antimicrobial action of LAB against the pathogens.Lb. plantarum 178; (ii) sausages inoculated with Lb. casei LC01; and (iii) uninoculated sausages.The experiments were performed as follows: (i) sausages inoculated with −1. The mixture was prepared according to the traditional formulation of sweet Calabrian salami—lean pork meat (69.5%), lard (17.5%), NaCl (2.2%), dextrose (0.4%), sodium ascorbate E301 (0.2%), fennel seeds (0.2%), sweet Eurodroga Calabra , bell pepper extract (8.6%), and freeze-dried strains (0.9%). The mixture (300 kg) was then stuffed into a natural casing—the weight of each sausage was 300–400 g. Sausages were ripened in an industrial plant as follows: stewing stage  99%); drying stage ; intermediate drying/ripening stage ; first ripening stage ; second ripening stage , and a final ripening/maturation stage .The strains had been previously freeze-dried, and the viable count was ca. 10 log cfu·gFor microbiological analysis, the casing was discarded and 25 g of the inner part was randomly selected, diluted with sterile saline solution (0.9% NaCl) (1:10), and homogenized using a Stomacher LAB Blender 400 . The homogenates were serially diluted and plated on selective media and incubated under appropriate conditions.−1)  incubated at 30 °C for 72 h under anaerobiosis for LAB; Sabouraud Dextrose Agar + chloramphenicol (0.1 g·L−1)  incubated at 28 °C for 48–72 h for yeasts; Malt Extract Agar (MEA) incubated at 25 °C for 4 days for molds; Pseudomonas Agar Base + CFC supplement for Pseudomonadaceae at 25 °C for 48–72 h; Mannitol Salt Agar for Staphylococci and micrococci at 37 °C for 24–48 h; Baird-Parker Agar Base for coagulase-positive staphylococci at 37 °C for 24–48 h ; and SPS Agar for sulphite-reducing Clostridia at 37 °C for 24 h under narrow anaerobic conditions. All media used were from Oxoid . In addition, MacConkey MUG Agar for E. coli at 37 °C for 24–48 h, X.L.T. Agar for Salmonella sp. for 24–48 h, and Listeria Palcam Agar for L. monocytogenes at 37 °C for 24–48 h were used. All media used for these pathogens were from Liofilchem . The results for plate counts were confirmed by microscopic observations.The media and the conditions used were: Plate Count Agar  incubated at 30 °C for 24–48 h for mesophilic bacteria; Violet Red Bile Glucose Agar (VRBGA) incubated at 37 °C for 18–24 h for Enterobacteriaceae; MRS agar + cycloheximide (0.17g·L(a)The pH was measured on sausage homogenate through a pH meter Crison 2001 .(b)Color was monitored by colorimetric measurements using a Tristimulus Colorimeter Chromameter-2 Reflectance , equipped with a CR-300 measuring head. The instrument was standardized against a white tile before each determination. The color of the sausages was determined by a Hunter scale as L * (brightness), a * and b * (hue and saturation of the color). Data were the average of at least five repetitions.(c)Water activity measurements were performed by using a hygrometer AQUA LAB CX-2 .(d)Moisture content was measured by using Sartorius Thermal Balance , at 130 °C until the samples reached a constant weight.The following physico-chemical analyses were done:p < 0.05). Statistics was made through the software Statistica for Windows .The experiments were performed on at least two independent batches —for each batch, the analyses were made twice. Significant differences were pointed out through one-way analysis of variance or two-way analysis of variance (ANOVA) and Tukey’s test, as the post-hoc comparison test , as they are used as starter cultures to promote meat fermentation . HoweverCarnevali et al.  proposedLb. plantarum 178, with some confirmatory experiments done at lab level — the technological and functional characterization is proposed elsewhere [This research focuses on the third step  for the autochthonous strain lsewhere .Lb. plantarum 178, Lb. sakei ST2, and Lb. casei LC01) after 6 h   B. In addAcidification is a desirable technological characteristic, since it is a simple index with which to evaluate the performance of a starter. The production of organic acids during fermentation allows the reaching of a very low pH, preventing the growth of pathogens during ripening . MoreoveLb. plantarum 178 suggests that it is a robust microorganism able to start fermentation at low temperatures (15–18 °C) and perform an acidification kinetic at high temperatures (44 °C). This is an important trait because temperature is the limiting factor for the selection of starter cultures for sausages [Lb. plantarum 178 experienced acidification in a meat-simulating medium containing the highest concentrations of nitrates and nitrites allowed by current legislation and a concentration of NaCl 5% higher than the typical amount used in Italian sausages (2.5–4%) [The acidification, at 15 °C as well as at 44 °C, of sausages . Moreove(2.5–4%) ,21.S. aureus, L. monocytogenes, E. coli, and Salmonella sp. were investigated. Lb. plantarum 178 strain, since Lb. casei and Lb. sakei do not produce an inhibition halo. Lb. plantarum cultures always inhibited E. coli, Salmonella sp., and S. aureus, while L. monocytogenes was inhibited at 25 and 30 °C, but not at 15 °C. Similar trends were found with the supernatant, but not with the buffered supernatant. Babic et al. [Lb. plantarum 178 towards pathogens was mainly due to acidification.Another important characteristic to select a functional starter is the bioactivity toward pathogens. As suggested by Babic et al. , S. aurec et al.  reportedLb. plantarum 178 was validated at an industrial level and compared to a functional starter culture with similar traits (LC01). Uninoculated sausages experienced a slight decrease of pH from 5.6 to 5.3—lab counts were at 3–4 log cfu·g−1 for the whole fermentation, and Enterobacteriaceae attained a cell count of 7.3 log cfu·g−1 after 10 days. Humidity did not decrease (35–37%) and activity water (aw) was always at 0.95 (data not shown).As a final step, −1, which did not undergo significant changes during fermentation and ripening (21 days). In 178-inoculated sausages, the LAB count experienced a slight increase (from 7 to 8 log cfu·g−1).−1, immediately after mixing and sausage preparation, to 8.5 log cfu·g−1 after 14 days , they are used as starter cultures without the addition of LAB because their activity imparts to the sausage a “sweet” rather than a “sour” taste, which is preferred by consumers . However−1 at the beginning) decreased throughout ripening .Due to lactic fermentation, Enterobacteriaceae  to ca. 7.5 log cfu·g−1 after 14 days. On the other hand, the trend of molds was extremely variable, and a macroscopic examination suggested the predominance of Penicillium spp.Yeasts and molds played a key role during the last stages of ripening. w, and humidity—concerning color, hue angle values were always in the range of purple (data not shown).The evolution of fermentation and ripening was evaluated through color, pH, aw. The pH of both LC01 and 178-sausages showed the typical trend of sausage fermentation  [w also followed a typical trend, with a final value of 0.82 for 178-sausages and 0.87 for LC01-sausages at the end of fermentation (olisana) ,21,23 wientation B. In genw reduction, along with acidification, is a desirable trait—depending on the temperature, pathogen inhibition could be either the results of pH or aw [w reduction is more important than a decrease in pH [L. monocytogenes and Salmonella sp. inhibition is acidification [apH or aw . When thse in pH . At highfication .Lb. plantarum 178 , or 24% in the case of arum 178 C. These humidity ,23, and Lb. plantarum 178) at an industrial level and shows the possibility of performing the fermentation of traditional Italian sausage through functional starter cultures, combining the benefit of a controlled fermentation and possible health benefits due to the functional properties of the strain. From a practical point of view, Lb. plantarum showed some interesting technological properties: (i) a good acidification score at 25–30 °C, higher than the value found for two commercial strains; (ii) the ability to perform acidification at 15 and 44 °C; (iii) the ability to perform fermentation under real conditions, thus assuring a correct course for the physico-chemical parameters ; and good microbiological imprinting.The choice of an appropriate microorganism to improve the performance of a fermented meat matrix is very important—for an autochthonous strain, the transition from a laboratory to an industrial scale is a complex process, since the repeatability of the results is not guaranteed and many times strains that are promising at lab level cannot control an industrial process. This study represents the scaling up and the validation of a promising autochthonous strain (


Adequate B-vitamins concentrations in human milk are considered to be a prerequisite for healthy development of infants in early life. This study aims to determine the concentrations of B-vitamins in human milk from Chinese women and the relationships between their concentrations and different geographical origin, lactation stages, socioeconomic characteristics, and dietary intake.n = 150), Suzhou (n = 146), and Guangzhou (n = 147) cities. Thiamine, riboflavin, vitamin B3 (nicotinamide and nicotinic acid), and vitamin B6  in human milk were analyzed by high performance liquid chromatography-tandem mass spectrometry. Pantothenic acid, biotin, and folates in human milk were analyzed by microbiological assay. The information from one 24-h dietary recall and socioeconomic characteristics were collected by interview and structured questionnaire, respectively.Human milk was obtained from 443 healthy lactating women from Beijing ; riboflavin 20.8, 20.2, 11.9, 13.6, 15.6 (μg/100 g); vitamin B3 194.0, 300.0, 261.0, 212.5, 218.0 (μg/100 g); pantothenic acid 236.5, 291.0, 254.0, 179.0, 189.0 (μg/100 g); vitamin B6 6.34, 7.58, 8.60, 9.34, 10.20 (μg/100 g); biotin 0.462, 0.834, 0.606, 0.523, 0.464 (μg/100 g); folates 0.730, 2.390, 2.440, 2.420, 2.330 (μg/100 g). The levels of B-vitamins presented regional differences and varied significantly among different lactation stages. The inversely associations of thiamine, vitamin B6, and folates with maternal BMI were found in multivariate analyses (p < 0.05), as well as higher pantothenic acid, folates, and biotin concentrations in lactating women with supplement intake when compared with those without (p < 0.05). Riboflavin concentrations associated with regular exercise was found in multivariate analyses (p < 0.05).B-vitamins concentrations in human milk varied greatly among individuals. The median concentrations of B-vitamins of The present study indicated regional and socioeconomic factors, lactation stage, and supplement intake may influence B-vitamins concentrations of human milk in healthy Chinese mothers. Further studies on accurate and complete analysis of all vitamin forms are crucial for giving a more comprehensive understanding of vitamin status in human milk.NCT01971671. Registered 13 October 2013.ClinicalTrials.gov, The online version of this article (doi:10.1186/s40795-017-0139-1) contains supplementary material, which is available to authorized users. According to the global strategy for Infant and Young Child Feeding from World Health Organization, exclusive breastfeeding is recommended during the first 6 months of life to ensure an optimal growth, development, and health of the infant , 2. Ther6, biotin, and folates are essential nutrients for maternal health during pregnancy and lactation and particularly important for the growth of infant , riboflavin-, nicotinic acid-d4, nicotinamide-d4, pyridoxine-d2, pyridoxal-[2H3] and pyridoxamine-[2H3] at a concentration of 200 μg/L) were added. Sample analysis was performed on a HPLC-MS/MS system. Liquid chromatography (LC) was performed using an Agilent 1100  LC system. Chromatographic separation was carried out with a Waters Acquity HSS T3 column . Column temperature was set to 30 °C and the autosampler remained stable at 15 °C. Ammonium formate aqueous solution  and acetonitrile (solvent B) served as mobile phase at a flow rate of 0.6 mL/min. The gradient process was: 0 min, 75% A; 1 min, 75% A; 1.1–10 min, 50% A; 10.1–15 min, 50% A; 15.1–16.5 min, 75% A; 16.6–0 min, 75% A. An API 4000 triple quadrupole mass spectrometer  was used for detection of the chromatographic separation. Nestle Internal reference material (bovine milk-based infant formula) was used for method validation and quality control (QC) sample as well, reference values were established by Nestle Proficiency Test participated by Nestle laboratories and third party laboratories. Coefficient of variation of repeatability is ranged from 3.7 to 13% and Coefficient of variation of intermediate reproducibility is ranged from 5.4 to 12%. The internal reference samples were regularly included and analyzed in duplicate during analytical runs. The limit of detection (LOD) for thiamine, riboflavin, vitamin B3 and vitamin B6 were 0.65, 1, 1, and 0.65 (μg/100 g), respectively. Recovery rates of vitamin B1, vitamin B2, vitamin B3, and vitamin B6 were ranged from 92 to 107%. Cross-talking in scheduled multiple reaction monitoring (MRM) was not observed between internal standards and analytes. The result of Vitamin B3 is the sum of nicotinamide and nicotinic acid, and it is expressed as nicotinic acid (niacin). As the mol masses are nearly identical both masses are only summed up. The result for Vitamin B6 is reported as pyridoxine. Thus pyridoxal hydrochloride (correction factor 0.831) and pyridoxamine dihydrochloride (correction factor 0.702) are converted to pyridoxine.One gram of sample was weighted in 15 mL centrifuge tube and hydrolyzed with 0.5 mL of hydrochloric acid (1 M) in autoclave for 30 min at 120 °C. Samples were cooled down and pH was then adjusted to 4.5 ± 0.5 by addition of HCl or NaOH. 0.01 g of taka-diastase was added to the samples, placed in water bath at 45 °C for 3 h. Sample volume was then adjusted to 10 mL with water and filtrated through 0.45 μm filter. 1 mL of sample extract was taken and isotopic labeled internal standards . After having applied all kit instructions including microorganism incubation at 37 °C in the dark for 20–24 h (pantothenic acid) or 44–48 h (biotin and folates), the turbidity of each vial was measured with a microtiter plate reader at 610–630 nm , for the calculation of pantothenic acid, biotin, and folates concentration. The LOD values for pantothenic acid, biotin, and folates were 0.074, 0.080, and 0.160 (μg/100 g), respectively. The measurements of QC samples described above  at predetermined intervals were regularly performed to obtain QC curve for the demonstration of method’s reliability. Recovery rates of pantothenic acid, biotin, and folates were ranged from 93 to 112%.One gram of sample was weighed and diluted with deionized water (40 mL). Sample extraction was then performed in a water bath at 95 °C for 30 min. After centrifugation , sample extracts could be further diluted with sterile water provided from the test kit if needed. Sample extracts were then pipetted on the commercially available microtiter plate VitaFastExperienced and trained researchers conducting dietary interviews obtained the information of dietary recall during the previous 24-h from participants by face-to-face interview when milk samples were collected. All of the foods intakes were coded and B-vitamins  were analyzed using a database according to Chinese Food Composition (CFC) tables 2004 & 2009 consist of 1773 food items , 38. Conpostpartum, 12–30 d postpartum, 31–60 d postpartum, 61–120 d postpartum, and 121–240 d postpartum). Before the progress of analysis about B-vitamins, Shapiro-Wilk test was employed to determine whether B-vitamins in human milk had a normal distribution or not. Because of non-normal distribution in human milk vitamins, naturally logarithmic transformations were applied when doing analysis of variance (ANOVA) according to research cities and lactating stages. Multivariate linear regression analysis was used to describe the relationship between B-vitamins concentrations in breast milk (dependent variables) and socioeconomic characteristics (independent variable) of lactation women. A stepwise forward selection process was used in which the independent variables and confounders  were added to the models according to their significance . Homogeneity and bias was showed in the analyses of the residuals of the final models. The associations between B-vitamins concentrations and diet characteristics such as dietary B-vitamins intakes were evaluated by partial-correlations adjusted for research cities and lactation stages. All statistical analyses were performed by using the SPSS software (Ver. 20.0) , and the level of significance was set at p < 0.05 based on a two-sided calculation.Socioeconomic characteristics of lactating women were described as count (percentage) for categorical variables and median value with interquartile range for continuous variables without normal distribution. Chi-squared tests  and Kruskal-Wallis tests (continuous variables) were used to compare participants’ characteristic according to stages of lactating period. Median (interquartile range) and mean ± standard deviation were calculated for each of B-vitamin according to research cities  and stages of lactation  (B1 vitamer), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) (B2 vitamer) and pyridoxal 5’-phosphate (PLP) (B6 vitamer) Therefore, these vitamin concentration are likely to be underestimated.The selected methodologies in this study allowed us the quantification of thiamine as B6 and folates increased progressively with duration of lactation (p for trend < 0.001). In contrast, riboflavin and pantothenic acid concentrations fell over time (p for trend < 0.01). Unlike the other vitamins, the levels of vitamin B3 and biotin remained almost constant as lactation stage increased (p for trend > 0.05).The concentrations of B-vitamins in different stages of lactation are shown in Table p < 0.05) except for biotin in human milk. The concentrations of thiamine and riboflavin were significantly higher in human milk from Beijing city than those from Suzhou and Guangzhou cities (p < 0.001). The concentrations of vitamin B3 and vitamin B6 were significantly lower in human milk from Suzhou city than the other two cities (p < 0.05). The level of pantothenic acid was significantly higher in human milk from Suzhou city than those from Guangzhou (p < 0.05). Meanwhile, the folates content in human milk from Suzhou city was significantly higher than that from Beijing city (p < 0.001).The concentrations of B-vitamins in human milk from Chinese mothers living in Beijing, Suzhou, and Guangzhou cities are shown in Table p > 0.05). The adjusted associations between B-vitamin concentrations in human milk and the socio-economic characteristics of lactating women are summarized in Table p < 0.05). Additionally, compared with lactating women with normal BMI, lower vitamin B6 in those overweight and obesity women and higher folates in those underweight women were found in this study . After adjustment for potentially confounding factors, significantly higher pantothenic acid, biotin, and folates were observed in lactating women with dietary supplement intake when compared with the other women . Meanwhile, the concentrations of riboflavin in human milk from the lactating women with regular exercise were significantly higher than those women without (p < 0.05).No significant correlations were observed between B-vitamins and maternal age, educational level, family income, and delivery mode in single factor analysis .In partial-analyses, after adjustment for investigated cities and lactation stages, no significant correlations were observed between vitamins in human milk and the corresponding vitamins in dietary intakes (3 concentrations were found in this study when compared with other previous studies in China [1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (nicotinamide and nicotinic acid), and Vitamin B6  so that the contents of these vitamins analyzed by HPLC-MS/MS methods were much lower than the microbiological assays.When compared with previously reported results of investigations into human milk of Chinese lactating women, the same vitamers were analyzed and our results were generally comparable –14. Thouin China , 14, ourin China . Generalin China . HPLC-MS6  concentrations increased with lactation stage increasing, which were consistent with our results. The finding from Ford et al. [6 increased progressively with duration of lactation, our results of only part of the total vitamin contents within these trends. However, riboflavin and pantothenic acid concentrations decreased as the lactation stage increasing in the present study, which are contradictory with the previous studies [postpartum 8–14 d) and then decreased with lactation stage increasing, which are consistent with our results. With lactation stage increasing, folic acid concentrations increased, which are similar to the trend of Ford et al. [Ren et al.  reportedd et al.  and Sakud et al.  showed t studies , 40. The studies  showed t1), Beijing had higher proportion of other cereals such as wheat and millet. This may explain the higher concentration of Thiamine in Beijing. Furthermore it is well known Riboflavin is widespread throughout many kinds of foods, especially from animal sources. Higher levels of riboflavin in human milk from Beijing city may be due to higher milk and eggs intake than those from Suzhou and Guangzhou cities. . According to the results from 24 h dietary recall, highest intakes of folates were found in lactating women from Suzhou compared to others , and it was expected that folic acid were higher in human milk from Suzhou city. Actually, regional differences of water-soluble vitamins in human milk including between rural and urban area [Remarkably, dietary intakes and supplementations may play core roles in the levels of B-vitamins in human milk , 16–19. ban area , 14, betban area  had been6, and folates, were found in the present study. High postpartum weight retention is a strong independent risk factor for several chronic diseases such as obesity, cardiovascular disease, and type 2 diabetes [6, biotin, and folates concentrations. Indeed, in this study, the levels of pantothenic acid, biotin, and folates found in human milk from women using vitamin supplement were markedly higher than those without, and these findings agreed with previous studies [According to the previous studies , 44, matdiabetes , thus ladiabetes . Furtherdiabetes , 20, 46  studies –20 on th6 intake on the content of the vitamin in milk from 19 healthy subjects, and found significant correlation  between them. Similarly, Johnston et al. [r = 0.65, p < 0.01) between pantothenic acid in the diet of the mother the day preceding milk collection and the pantothenic acid content of the milk. Results of this study could not confirm this due to likely a limitation of the dietary intake assessment method [Mother’s B-vitamins intake has been found to clearly correlate with their concentrations in human milk , 16–19. n et al.  found a t method  which onAlthough state-of-the-art and/or reference analytical methodologies were applied, caution should be taken when trying to make conclusions about absolute vitamin content in human milk from Chinese mothers. Since some metabolites could not be estimated, only partial information was given on some vitamins.In conclusion, our results of B-vitamin concentrations in human milk generally agree with previously reported studies conducted in China. Our findings suggest that some regional, lactation stage, socio-economic factors including maternal BMI, regular exercise, and dietary supplement may have effects on B-vitamins in human milk from healthy Chinese mothers. In view of adequate B-vitamins concentrations in human milk as a prerequisite for healthy development in early life, further studies on accurate and complete analysis of all vitamin forms (vitamers/metabolites) are crucial for giving a more comprehensive picture of vitamins concentrations and evolution in human milk.Additional file 1:Table S1. Multivariate linear regression models considering riboflavin and biotin concentrations in human milk after the ln transformation as the dependent variables and the other variables studied as independent variables. After adjustment for potentially confounding factors, significantly higher biotin were observed in lactating women with dietary supplement intake when compared with the other women (p < 0.05). Meanwhile, the concentrations of riboflavin in human milk from the lactating women with regular exercise were significantly higher than those women without (p < 0.05). (DOCX 16 kb)


Lactobacillus salivarius LS358 and L. rhamnosus LR753, two type strains L. salivarius ATCC 11741T and L. rhamnosus ATCC 7469T. The ensiling temperatures were designed at 30 °C and 45 °C, and the sampling took place after 0, 3, 7, 14, and 60 days of fermentation. The higher pH and dry matter losses were observed in the silages stored at 45 °C compared to those stored at 30 °C. Silages inoculated with strains LS358 and LR753 at 30 °C had a lower ratio of lactic acid/acetic acid. The dominant bacterial genera gradually changed from Pediococcus and Lactobacillus to Lactobacillus in silages during ensiling at 30 °C, while the bacterial community became more complex and fragmented after 7 d of ensiling at 45 °C. The high temperatures significantly led to a transformation of the LAB population from homo-fermentation to hetero-fermentation. This study is the first to describe microbial population dynamics response to high temperature during corn ensiling, and the results indicate that L. rhamnosus 753 shows potential ability to improve silage fermentation in tropics and subtropics.To develop a silage fermentation technique to adapt to global climate changes, the microbiome and fermentation dynamics of corn silage inoculated with heat-resistant lactic acid bacteria (LAB) under high-temperature conditions were studied. Corn was ensiled in laboratory silo, with and without two selected strains,  Silage is the world’s important feed resource for ruminants . Whole-pDue to the effects of global warming, extremely high temperatures have become a key factor affecting the quality of silage by accelerating the fermentation process and aerobic deterioration in tropical and subtropical regions . It is gLactobacillus plantarum can inhibit the growth of microbe in silage by accelerating the decrease of pH [L. buchneri as most used heterofermentative LAB strain can produce acetic acid to inhibit the growth of yeast and mold, thus improving aerobic silage stability [L. plantarum and Pediococcus acidilactici) had no positive effect on Ryegrass silage at 45 °C. The effect of inoculating commercial LAB (L. plantarum MTD-1) was also limited in Napier grass silage, which stored at 50 °C [L. plantarum not only failed to improve the silage quality but also accelerated the aerobic deterioration in corn silage stored at 35–40 °C. These studies indicate that commercial LAB, which mostly screened from temperate regions, have limited adaptability to high-temperature environments.Generally, to ensure that silage enters the fermentation stage dominated by LAB as soon as possible, LAB inoculants are commonly used in silage production . Typicalse of pH , while Ltability . Howevertability , which wtability ,8,9, thetability  reportedat 50 °C . Guan etat 50 °C  found thSilage is a fermentation process dominated and driven by microorganisms . ObviousL. salivarius 358 and L. rhamnosus 753) in a hot environment (30 °C and 45 °C).To develop a silage fermentation technique in response to global warming, the current study aimed to apply NGS to determine the dynamic  changes of microbial community and fermentation profile of corn silage prepared with two screened heat-resistant LAB (Zea mays L. cultivar Yayu No.8) used in this experiment was harvested at the kernel half milk line on 27 June 2018, at the Farm of Sichuan Agricultural University in Chongzhou, Sichuan Province, China (N30°33′23.98″ E103°38′42.61″). The self-propelled forage harvester  was set to chop at a theoretical length of 15 mm. The freshly chopped, whole-crop corn used in this study contained 283.8 g kg−1 dry matter (DM), and the contents of the crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and WSC were 59.31, 451.59, 222.39, and 177.78 g kg−1 DM, respectively. Two strains L. salivarius 358 (LS358) and L. rhamnosus 753 (LR753) were isolated from forage crops and silages in hot and humid areas of southwest China . Both strains can grow at high-temperature and low pH conditions and produce more lactic acid than other inoculant strains and isolates in silage environments. According to the screening results of physiological and biochemical properties and small-scale silage fermentation tests of more than 200 LAB isolates, the LAB strains were selected for improving silage fermentation and inhibiting aerobic deterioration of silage [L. rhamnosus 753 (LR753) and L. salivarius 358 (LS358), two type strains L. salivarius ATCC 11741 (LS) and L. rhamnosus ATCC 7469 (LR). Four LAB strains were inoculated as a single culture by using de Man, Rogosa and Sharpe (MRS) broth  and distilled to an equivalent of around 106 colony-forming units cfu g−1 of fresh matter (FM), then sprayed evenly on the corn material and thoroughly mixed. One kg of the mixed material was filled in a sterile plastic bag  and evacuated and sealed by an internal pumping vacuum packaging machine . Each treatment had three biological replicates, a total of 120 vacuum-bags were separated and stored in a dark environment at 30 °C and 45 °C, respectively. The fresh corn was taken for analysis of chemical composition and microbial community. Samples of 3, 7, 14, 60 d for ensiling were used for the analysis of the dynamic changes of microbial community and fermentation, and a sample from the 60 d was also used for the analysis of chemical composition. After opening all samples, the number of microorganisms was detected immediately, and two subsamples from each sample were taken quickly into −80 °C lab freezers  for DNA extraction and detection of relevant fermentation parameters.The whole plant corn  and passed through a 1 mm mesh sieve for future chemical analysis. An Ankom 200 system  was used to test NDF and ADF, and the test method was in accordance with the instructions. The CP was determined by the Kjeldahl method and WSC was measured by the thracenone-sulfuric acid method [Three fresh samples and all 60-day silage samples were placed in an air-circulated oven at 65 °C for 72 h to test the DM. The weight and DM of the samples before and after 60 d of silage were used to calculate DM recovery (DMR). The dried samples were ground by grinder  were analyzed by high-performance liquid chromatography (HPLC) with a UV detector (210 nm) and a column , according to the method by Guan, et al. [3-N was determined according to the method by Weatherburn [A 20 g of wet silage samples from 3, 7, 14, and 60 d were added to 180 mL of distilled water and stayed overnight in a refrigerator at 4 °C, and then filtered through two layers of cheesecloth to obtain the silage extract. The pH of the extract was tested using a pH meter , and the remaining samples were used to test organic acids concentration and NHtherburn .The amount of LAB was quantified by De Man, Rogosa, Sharpe agar , molds and yeasts counts were cultured on Potato Dextrose Agar , and coliform bacteria were quantified using Violet Red Bile Agar .The method of DNA extraction referred to Guan et al.  and Yan 16S rRNA genes of distinct regions (16S V4) were amplified used the specific primers 515F (5′-GTTTCGGT GCCAGCMGCCGCGGTAA-3′) and 806R (5′-GCCAA TGGACTACHVGGGTWTCTAAT-3′) [Next-generation sequencing reads were assembled using FLASH (V1.2.7) . Accordi−1 and were log-transformed prior to statistics. The statistical analyses were performed using the GLM procedure of SPSS v19.0. Tukey’s honest significant difference (HSD) test was employed, and significance was declared at p < 0.05.Microbe populations were estimated as colony-forming units (cfu) gp < 0.05), LS-treated silage had the highest pH at 45 °C, while LR-treated silage had the lowest pH at 30 °C (p < 0.05). Control silages at both temperatures had the highest NH3-N (g kg−1 TN) rates compared to the LAB inoculated silages (p < 0.05). Counts of LAB in all silages increased at 30 °C with large amounts of lactic acid production after three days of fermentation (p < 0.05). However, although high contents of lactic acid were found in all silages at 45 °C, lower LAB counts were observed compared to forage. The number of yeasts, molds, and coliforms decreased in all silages at both temperatures compared to forage, and there was no acetic acid produced after 3 d of fermentation. Temperature, inoculants, and their interaction had an extremely significant effect on pH, number of LAB, and yeast (p < 0.01).Fermentation characteristics and microbial counts of corn silage at different periods are shown in p < 0.05), the highest pH was found in the control silage at 45 °C (p < 0.05). NH3-N contents in all silages increased compared to silages ensiled for 3 d and ranged from 20.24 to 27.40 g kg−1 TN. Although the pH value of all silages did not change much, lactic acid contents increased in all silages compared to silages ensiled for 3 d. Acetic acid in all silages at 30 °C were observed after ensiling for 7 d, while only screened LAB (LS358 and LR753) inoculated silages observed acetic acid (AA), and the highest AA contents were found in LR753-inoculated silage stored at 30 °C (p < 0.05). LAB counts of controls at 30 °C increased while LAB-treated silages showed small decline compared to silages ensiled for 3 d. LAB counts of all silages at 45 °C still showed a downward trend. Mold was detected only in control silage at 45 °C, which was 4.54 log10 cfu g−1 of FM. Temperature, inoculants, and their interaction had an extremely significant effect on pH, AA contents, LA/AA, number of LAB, and yeast (p < 0.01).After 7 d of fermentation , all sil3-N contents with the control at 30 °C (p < 0.05). NH3-N (g kg−1 TN) of all groups increased with the extension of the fermentation process. The content of LA did not change much compared to the silages ensiled for 7 d, except for the LR-treated silage at 30 °C that increased significantly, and was found to be the highest (p < 0.05). The content of AA continued to increase, and the highest AA content appeared in LR753 treatments at 30 °C (p < 0.05), which reached 61.22 g kg−1 of DM. The number of LAB in all 30 °C treatments remained between 2.71 and 7.09 log10 cfu g−1 of FM, the highest number of LAB were observed in LR753 treatments at both temperatures. The number of yeasts continued to decrease, and there were still no mold and coliforms detected in all silages. Temperature, inoculants, and their interaction had an extremely significant effect (p < 0.01) on pH, AA contents, number of LAB, and yeast. As the fermentation time prolonged to 14 d, as shown in p < 0.05). The NH3-N (g kg−1 TN) continued to increase, but all silages did not exceed 50 g kg−1TN, the lowest NH3-N (g kg−1 TN) was observed in LR753 groups at 45 °C (p < 0.05). The contents of NH3-N (g kg−1 TN) and LA in all silages at 45 °C were higher than the same-treated silages at 30 °C, while silages at 30 °C had higher AA contents (p < 0.05). The number of LAB decreased in all groups at 30 °C compare to silages ensiled for 14 d, ranging from 4.15 to 4.66 log10 cfu g−1 of FM. There were no LAB, yeasts, mold, and coliforms detected in all silages at 45 °C. Temperature, inoculants, and their interaction had an extremely significant effect on pH, NH3-N (g kg−1 TN), and LA/AA (p < 0.01).At the 60 d of ending, the pH of all samples at both temperatures ranged from 3.81 to 4.06 , the higp < 0.05) higher than that at 45 °C. CP contents of all silages increased compared to forage except for LS treatment at 45 °C. Contents of WSC decreased after 60 d of fermentation; however, silages at 45 °C had significantly (p < 0.05) higher WSC contents than silages at 30 °C. The NDF and ADF showed a small difference in all treatments at both temperatures. Temperature, inoculants, and their interaction had an extremely significant (p < 0.01) effect on DM, CP, and WSC.Chemical composition of corn silage ensiled for 60 d at 30 °C and 45 °C are shown in Leuconostoc, Klebsiella and Lactococcus spp. were the dominate LAB in fresh corn forage. The abundance of Lactobacillus and Pediococcus spp. increased significantly after 3 d of fermentation. Pediococci in silages at 45 °C was generally higher than that at 30 °C. Especially, LS358 and LR753treatments had a higher abundance of pediococci at both temperatures. The communities of bacteria in all silages at 30 °C ensiled for 7 d had a similar situation with that ensiled for 3 d, except for the abundance of lactobacilli, which increased a lot while pediococci decreased in LR753 treatments at 30 °C. However, the bacterial community dramatically changed compared to the 3 d of fermentation at 45 °C. The abundance of pediococci sharply decreased while a large number of bacteria with small abundance began to appear in silages ensiled for 7 d at 45 °C. Besides, Bacillus spp. presented in the control group and samples inoculated with LS358, and Acetobacter spp. was found in LS and LR-treated groups at 45 °C. As the fermentation time prolonged to 14 d, pediococci was still the dominate bacteria in the LS358-treated group, while the LR753 group had the highest abundance of lactobacilli at 30 °C. Novelty, a high abundance of acetobacter was found in all silages except for the LR753 treatment at 45 °C. At the ending of fermentation (60 d), lactobacilli were the prevalent bacteria in all silages at 30 °C, followed by acetobacter, while the highest abundance of lactobacilli was observed in LR753 and LR treatments. The bacterial communities of each group were more complex at 45 °C, the abundance of lactobacilli and acetobacter decreased compared to silages ensiled for 14 d, while the abundance of Pectobaterium, Brevundimonas, and Bosea spp. increased a lot. Besides, the Bacillus spp. became the dominant bacteria in LR753 treatment at 45 °C.The taxonomic profile of the bacterial core microbiome varied among fresh forage and dynamic changes of corn silages at 30 °C and 45 °C are shown in p < 0.05) affecting the bacterial community in corn silage from 3 d to 60 d, of which the greatest effect was observed at 60 d . The significant (p < 0.05) effect (p < 0.05) of the inoculants on the bacterial community only lasted to d 7, and there was no significant effect (p > 0.05) at 14 and 60 d of ensiling. Moreover, the R-value < 0 of the inoculants was observed at 7, 14, and 60 d of ensiling.As shown in In order to investigate the effects of temperature on the dynamic change of microbial community in corn silages, we pooled the samples under the same temperature and fermentation time into one group . There wWilkinson and Muck  reported3-N (g kg−1TN) in the initial stage of the ensiling of all silage at 45 °C were higher than those at 30 °C, while the number of LAB was lower. After 60 d of fermentation, no LAB was observed at the detected level in all silages; this indicated that most LAB could adapt to 45 °C at the beginning of ensiling, but they could not survive after two weeks under such high temperature. Clostridia generally have a higher optimum growth temperature, typically 37 °C, and will grow at 45 °C [3-N (g kg−1 TN) of all treatments at both temperatures increased, especially at 45 °C. The optimal growth temperature of Clostridium is usually higher than that of LAB, and it can grow normally at 45 °C [L. buchneri was reported to improve aerobic stability of silage [L. buchneri was screened from temperate regions [McDonald et al.  indicateat 45 °C ; therefoat 45 °C ,24. In tat 45 °C , while tat 45 °C , the temat 45 °C , the ratf silage , many exf silage ,28,29 exf silage  and dosef silage ,33. Due  regions . In the Generally, the information obtained through the detection of these conventional fermentation data was very limited, so a deeper understanding of the dynamics of microbial communities during ensiling is highly required.Weissella, Pseudomonas, Lactobacillus, and Leuconostoc spp. were the most dominate epiphytic bacteria genera, while Leuconostoc, Klebsiella, and Lactococcus spp. were the most prevalent bacteria found in the current study. These results indicated that the epiphytic bacterial community was not only affected by forage and climate type, but it was more related to individual differences, and although Lactobacillus and Lactococcus spp. existed in fresh corn, they were usually not dominant bacteria. In this study, Lactobacillus and Pediococcus spp. began to dominate at 3 d of the ensiling, this was different from the report by Drouin et al. [Lactobacillus spp. began to dominate at 14 d of fermentation in corn silage inoculated with L. buchneri and L. hilgardii. This may be because the higher temperature in the current study promoted the rapid propagation of LAB, which is consistent with the results reported by Keshri et al. [Lactobacillus spp. began to dominate at 2 d of corn silage in the warm climate. Lactobacillus and Pediococcus spp. are common predominant bacteria genera in silage [Generally, epiphytic bacterial communities are highly related to forage types  and climn et al. , which Li et al.  that Lacn silage . EspeciaAcetobacter species was not common in the reported laboratory small-scale silage, while it was regularly detected in bunker-made corn silage [L. rhamnosus has been reported as a silage additive, especially as heat-resistant inoculants [L. rhamnosus was more effective than other inoculants at 30 °C. Results from Anosim and PERMANOVA analysis showed the same finding, the temperature was the main factor affecting the bacterial community during ensiling, and achieved the greatest effect on the terminal of fermentation. While the R-value < 0 of the inoculants was observed at 14 and 60 d of ensiling, indicating that inoculants affected the bacterial community only lasted in the early two weeks.Higher ensiling temperatures typically lead to a shift from homofermentative to heterfermentative LAB population . Currentn silage . Howevern silage . In receoculants ,36. Howe, et al. [Higher ensiling temperatures typically lead to a shift from a homofermentative to a heterofermentative LAB community , which o, et al.  and Kim , et al. . Wilson , et al.  have rep, et al. , thus inLeuconostoc, Klebsiella, and Lactococcus spp. Lactobacilli were the prevalent bacteria in all silages at 30 °C, followed by acetobacter, while the bacterial communities became more complex at 45 °C at 60 d of ensiling. High temperature affected the dynamic changes of microorganisms in corn silage and resulted in a shift from homofermentative to heterofermentative LAB community, which led to poor fermentation with more DM loss and higher NH3-N content. The heat-resistant strain L. rhamnosus 753 showed the potential possibility to respond to global warming for improving silage fermentation in tropics and subtropics. The bacterial monitoring dynamics by NGS technology is expected to better understand the relationships between microbial community and silage fermentation.The bacterial community of fresh whole-crop corn was found to be dominated by


Lyngbya species’ characterization is limited in the literature, we characterize this cyanobacterium’s growth and biomass. L. purpureum was grown and analyzed under different salinities, culture media, and incubation times to determine the best conditions that favor its cell growth and the general production of proteins, carbohydrates, lipids, and some pigments as phycocyanin and chlorophyll a. In this study, each analyzed biomolecule’s highest content was proteins 431.69 mg g−1, carbohydrates 301.45 mg g−1, lipids 131.5 mg g−1, chlorophyll a 4.09 mg g−1, and phycocyanin 40.4 mg g−1. These results can provide a general context of the possible uses that can be given to biomass and give an opening to investigate possible biocompounds or bio metabolites that can be obtained from it.Cyanobacteria are essential for the vast number of compounds they produce and the possible applications in the pharmaceutical, cosmetical, and food industries. As  Lyngbya is an excellent example of cyanobacterial strains, showing extraordinary diversity in the production of more than 200 secondary metabolites with biological activity [Lyngbya strains appear to be halophilic, adapting to different salt concentrations in the medium. For instance, the strain Lyngbya contorta shows better cell growth and protein production while increasing the salt concentration from 5 ppt to 40 ppt [Currently, cyanobacteria have risen in importance due to the many compounds produced by them and their possible applications in the food, energy, and pharmaceutical industries. The genus activity ,2. Some o 40 ppt ,4.Lyngbya strains can produce pigments and bio-compounds with potential applications as pharmaceuticals due to their properties, including neuroprotective, anti-oxidant, antibacterial, antifungal, anti-inflammatory, antileishmanial, antidiabetic, and anticancer activity . Regarding BG11 medium  and in BG11 in medium salt concentration (301.45 mg g−1)  . The ave4 mg g−1 a,b. FromL. purpurem is chlorophyll a  is essential for chlorophyll a production. For Bold 3N cultures, it was seen that low and medium salt concentrations enhance chlorophyll a content at day 25 and 15, respectively.One of the most common pigments produced by ophyll a . The higLyngbya sp. produces phycobiliproteins as the phycocyanin [−1 to 40.4 mg g−1, whereas with BG11, phycocyanin ranged from 6.2 mg g−1 to 23.7 mg g−1 . This amount of nitrogen and the inorganic carbon source can positively affect the growth of the microalgae. Therefore, better growth of L. purpurem was obtained using the BG11 culture medium. The total dry biomass presented in this work resulted higher than the 0.642 g L−1 of Lyngbya’s biomass previously reported in the literature, where the BG11 medium did not contain any variation in the salt concentration [L. aestuarii in f/2 medium with a salinity of 0.03 ppb, producing 0.0024 g L−1 at day 30 . The biomass growth results in BG11 and Bold 3N at different salt concentrations give the possibility of using these media to obtain an optimal growth of L. purpurem and marine Lyngbya species. Compared to other cyanobacteria, the obtained biomass of L. purpureum can be compared with the 1.45 g L−1 and 1.63 g L−1 obtained by Spirulina subsalsa BDU51591 and Oscillatoria sp. BDU51731, respectively [BG11 medium contains an inorganic carbon source  protect cyanobacteria against osmotic and salt stress [Furthermore, cyanobacteria production in seawater can be an excellent option to produce biofuels and metabolites of interest . Also, tt stress . Therefo−1 and 0.75 g L−1, respectively). It has been reported before that a higher level of nitrogen stimulates the production of proteins since nitrogen is one of the principal components in amino acids [It can be observed that independently of the salt concentration evaluated; there was a higher protein production in the BG11 culture medium than in Bold 3N medium. The high amount of protein produced may result from the nitrogen concentration used in BG11, which was twice the concentration used in the Bold 3N medium  compared to the produced by Chlorella sp. (85mg g−1) and Ankistrodesmus sp. (81 mg g−1) grown in the BG11 culture medium.Regarding the total production of lipids, the highest concentration was obtained using the Bold 3N medium at the low salt concentration on days 20 and 25. Similar to our results, other studies have evaluated the response to nitrogen starvation in microalgae like ella sp. , Chlorelvulgaris , Scenedesmus sp. , and P. cornutum , findingntration . Also, oLyngbya sp. showed a lipid production of 102.95 mg g−1 at day 19 [L. dendrobia produced a total lipid content of 10.55% when it is cultured in BG11 medium. Similarly, L. limnetica, cultured in BG11, produced a lipidic content of 18.1% [L. aestuarii produced 14.4% DW of lipids when it was grown in f/2 medium [Lyngbya’s biomass, saturated and unsaturated FAME can be used as biofuels or for animal feed. For example, Chandra et al. reported palmitic and linoleic acid production. On the one hand, palmitic acid has antimicrobial properties; on the other hand, linolenic acid has been used as a dietary supplement. [Chroococcus sp. BDU20201 (5.79%), Spirulina subsalsa BDU51591 (18.51%), Oscillatoria salina BDU50701 (11.9%), and Oscillatoria sp. BDU51731 (16%), making this cyanobacterium a good option for obtaining biofuels. Our findings are also consistent with the results obtained with other Lyngbya strains, where 4–12% DW belongs to lipids [It is important to highlight that our lipids production was higher than most previous studies in the literature. For example, t day 19 , and L. of 18.1% , and L. 2 medium . Among tplement. ,65. Compo lipids .Lyngbya sp. For instance, the total carbohydrate content of 172.89 mg g−1 in Lyngbya sp. [−1 DW obtained in our study at day 25 in Bold 3N added with a high salt concentration. Also, L. aestuarii obtained an 18.4% of carbohydrates when it was grown in f/2 medium [L. purpurem in our study with BG11 medium . Large amounts of sucrose have been found in cyanobacteria; this carbohydrate is the most used for biofuels production. It was observed that the addition of optimal amounts of NaCl to enhance sucrose accumulation by Synechocystis sp. PCC6803. [Lyngbya purpurem can produce one of the highest carbohydrate contents among different microorganisms.Our results show a higher carbohydrate production in comparison to other strains of gbya sp.  was obta2 medium . This rePCC6803. . The totPCC6803. , which sa production has been reported in other cyanobacteria such as Spirulina platensis, finding a production of 4.28 mg g−1 [−1 [−1 [N. muscorum and P. foveolarum have been exposed to different salt concentrations where chlorophyll a achieved a production range from 14 mg g−1 to 20 mg g−1, approximately [a structure negatively in different cyanobacteria such as Spirulina platensis [The chlorophyll-8 mg g−1 , 11.5 mg g−1 [−1 , and 12. [−1 [−1 . These rximately . Furtherlatensis .Spirulina maxima’s phycocyanin production to answer stressful environmental conditions [It is interesting to observe that although a higher biomass growth and chlorophyll content were obtained in BG11 medium, in terms of phycobiliprotein production, the best culture medium was Bold 3N. From the factorial design analysis of Bold 3N cultures, it was determined that salt concentration is essential in phycocyanin production. Setyoningrum & Nur reported that an increase in salt concentration causes a significant increment in nditions . Table 2The reagents used for the culture medium, and the lipids and carbohydrates analysis were purchased from Sigma-Aldrich . The Lowry Protein Assay Kit was purchased from Thermo Fisher Scientific . The spectrophotometric measurements were taken in an absorbance microplate reader BGM Labtech .Lyngbya purpurem LB 2716 was purchased from UTEX  2·6 H2O 0.0494 mg L−1. The Bold 3N culture medium contained: NaNO3 0.75 g L−1, K2HPO4 75 mg L−1, CaCl2·2 H2O 25 mg L−1, MgSO4·7 H2O 75 mg L−1, KH2PO4 0.175 g L−1, Na2EDTA·2 H2O 4.5 mg L−1, FeCl3·6 H2O 0.582 mg L−1, MnCl2·4 H2O 0.246 mg L−1, ZnCl2 0.03 mg L−1, CoCl2·6 H2O 0.012 mg L−1, Na2MoO4·2 H2O 0.024 mg L−1.The strain TX, USA) . ErlenmeL. purpurem was evaluated in 2 different culture media (BG11 and Bold 3N) under 3 different salt concentrations: low (12.5 mg L−1), medium (25 mg L−1), high (50 mg L−1), during 3 different incubation time lengths .For each experiment, 0.30 g DW of wet biomass were used as seed culture and were inoculated into a 250 mL Erlenmeyer flask with the corresponding culture medium, resulting in 150 mL of total volume. A Factorial Design was used for this research and was applied to each set of experiment . Cell grL. purpurem. Its original composition contains 25 mg L−1 of NaCl, so this value was taken as a reference to set the different salinities used in this study. At incubation days 15, 20, and 25, the biomass of all salt concentrations  was dried at 4 °C for two days. The dry biomass (DB) was weighted and resuspended in 20 mL of bi-distilled water. The weight of DB was measured to obtain the cell growth behavior.The culture medium Bold 3N is often used to grow The DB was resuspended in 20 mL of bidistilled water and was used to quantify the proteins, lipids, carbohydrates and pigments present in the biomass. Each sample (20 mL) was sonicated in a Sonifier Cell Disruptor (Branson Model # 102 C) to achieve cellular lysis. After this, the biomass protein content was quantified using the Lowry Protein Assay Kit (Lot # RB230458A), the lipid content was quantified using the sulfo-phospho-vanillin assay described by Mishra et al.  and the a concentration was obtained by measuring the supernatant’s absorbance at 652 and 665 nm wavelengths by spectrophotometry. The concentration was calculated with the following formula described by [Each sonicated sample was also used to determine the pigments concentration. 5 mL of each sample were dried at 40 °C for two days, followed by a resuspension in 5 mL of methanol (95%) and incubation at 60 °C for 5 min. The samples were centrifuged for 10 min at 4000 rpm and 15 °C. The chlorophyll ribed by ,75:(1)ChTo evaluate the phycocyanin concentration, the DB was resuspended in 20 mL of bidistilled water after sonication. The phycocyanin concentration was obtained by measuring the supernatant’s absorbance at 592, 618, and 645 nm wavelengths by spectrophotometry. The concentration was calculated with the following formula described by :(2)PC mgn = 3) and expressed as +/− SD in Figures and Tables.All data were analyzed in Minitab 18 . A Multi-Factor ANOVA with a Tukey pairwise comparison per type of culture media was used in order to analyze the factorial design data. The coated values were taken from the triplicate samples , carbohydrate 301.45 mg g−1 , lipid 131.5 mg g−1 , chlorophyll a 4.09 mg g−1 , and phycocyanin 40.4 mg g−1 .This study reports the first characterization of the biomass of Lyngbya purpurem has shown to be an adequate protein and carbohydrates source, which can be used in the food industry as a food supplement for humans or animal feed. L. purpurem’s biomass has high nutritional value due to its protein, carbohydrate, and lipid content. Furthermore, it is important to highlight the antioxidant and anticancer properties conferred by chlorophyll a, namely the anti-inflamatory, anticancerous, hepatoprotective, and neuroprotective activities of phycocyanin. As a natural bio-factory, L. purpurem’s carbohydrates and lipids can also be used for biofuels and bioplastic production. Other applications, such as biofertilizer, nutraceutical, and biomaterials, can be evaluated as part of a future study to impact the pharmaceutical, nutraceutical, agricultural, and cosmetic industries.