Bioprocess development for endospore production by Bacillus coagulans using an optimized chemically defined medium.

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%-90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.

Sensors and Techniques for On-Line Determination of Cell Viability in Bioprocess Monitoring.

In recent years, the bioprocessing industry has experienced significant growth and is increasingly emerging as an important economic sector. Here, efficient process management and constant control of cellular growth are essential. Good product quality and yield can only be guaranteed with high cell density and high viability. Whereas the on-line measurement of physical and chemical process parameters has been common practice for many years, the on-line determination of viability remains a challenge and few commercial on-line measurement methods have been developed to date for determining viability in industrial bioprocesses. Thus, numerous studies have recently been conducted to develop sensors for on-line viability estimation, especially in the field of optical spectroscopic sensors, which will be the focus of this review. Spectroscopic sensors are versatile, on-line and mostly non-invasive. Especially in combination with bioinformatic data analysis, they offer great potential for industrial application. Known as soft sensors, they usually enable simultaneous estimation of multiple biological variables besides viability to be obtained from the same set of measurement data. However, the majority of the presented sensors are still in the research stage, and only a few are already commercially available.

Facilitated endospore detection for Bacillus spp. through automated algorithm-based image processing.

Bacillus spp. endospores are important dormant cell forms and are distributed widely in environmental samples. While these endospores can have important industrial value (e.g. use in animal feed as probiotics), they can also be pathogenic for humans and animals, emphasizing the need for effective endospore detection. Standard spore detection by colony forming units (CFU) is time-consuming, elaborate and prone to error. Manual spore detection by spore count in cell counting chambers via phase-contrast microscopy is less time-consuming. However, it requires a trained person to conduct. Thus, the development of a facilitated spore detection tool is necessary. This work presents two alternative quantification methods: first, a colorimetric assay for detecting the biomarker dipicolinic acid (DPA) adapted to modern needs and applied for Bacillus spp. and second, a model-based automated spore detection algorithm for spore count in phase-contrast microscopic pictures. This automated spore count tool advances manual spore detection in cell counting chambers, and does not require human overview after sample preparation. In conclusion, this developed model detected various Bacillus spp. endospores with a correctness of 85-89%, and allows an automation and time-saving of Bacillus endospore detection. In the laboratory routine, endospore detection and counting was achieved within 5-10 min, compared to up to 48 h with conventional methods. The DPA-assay on the other hand enabled very accurate spore detection by simple colorimetric measurement and can thus be applied as a reference method.

Laparoscopic Prophylactic Total Gastrectomy for Hereditary Diffuse Gastric Cancer in CDH1 Mutation Carriers.

Background: Patients with hereditary diffuse gastric cancer (HDGC) and germline mutations in the E-cadherin gene, CDH1, have a very high cumulative lifetime risk of developing diffuse gastric cancer. In these patients, it is formally recommended to perform a prophylactic total gastrectomy (PTG). Materials and Methods: We analyzed the course of patients with HDGC who have undergone PTG in our institution. Pedigree analysis, preoperative screening results, operative course, postoperative data, and complete stomach pathologic examination were performed in all patients. Results: Seven patients with confirmed CDH1 mutation underwent PTG, five were women, and average age was 27 years (range 17-42). Signet ring cell carcinoma was found in 1 patient in the preoperative surveillance endoscopic biopsies. Laparoscopic PTG was performed in all patients. There were two complications, an intestinal obstruction that required reintervention and an asymptomatic esophagojejunal anastomosis leak that resolved with conservative treatment. In all gastrectomy specimens, intramucosal signet ring cell carcinoma foci limited to the lamina propria were found (range 1-31), 83.5% were in the body-fundus region. The mean follow-up was 28.5 months (range 8-72). The mean weight loss was 9% (range 2-18). Postoperative symptoms associated with Dumping syndrome were the most frequent. All the patients reported of being very satisfied with the procedure and of having a better quality of life than expected before the procedure. Conclusion: Laparoscopic PTG is an excellent resource to prevent the development of advanced diffuse gastric cancer (DGC) in patients with HDGC with CDH1 mutation. The procedure was well tolerated with a high satisfaction rate and very good functional results. It should be considered in these patients due to the high risk of developing advanced DGC and the lack of effective and reliable surveillance studies.

Laparoscopic Heller Myotomy and Roux-en-Y Gastric Bypass as Treatment for Patients with Achalasia and Morbid Obesity: Outcomes in a Short Series of Patients.

Background: The association between morbid obesity and esophageal achalasia is very infrequent. However, over the last decade, these cases started to increase because of the disturbing rise of morbid obesity worldwide. Heller myotomy (HM) and laparoscopic fundoplication represent the best treatment option for esophageal achalasia. However, in morbidly obese patients with achalasia, the best treatment option is not established. We present laparoscopic HM and Roux-en-Y gastric bypass (RYGB) as an alternative treatment for morbidly obese patients with achalasia. Materials and Methods: We analyzed the course of patients with achalasia and morbid obesity in our institution undergoing a laparoscopic HM and RYGB, with at least 1 year of follow-up. Symptoms questionnaire, body mass index (BMI), and minuted esophagogram before and after treatment were performed in all patients. Results: Seven patients underwent laparoscopic HM and RYGB. All patients had dysphagia. The mean BMI before the onset of symptoms was 42 kg/m2 (range 40 to 50). In 5 patients, preoperative contrast esophagram showed mild or marked dilated esophagus, and in 2, one esophageal curve and severe dilation. Manometry confirmed the diagnosis. At a mean follow-up of 38 months (range 14-69), all patients reported a marked improvement in dysphagia, with median overall satisfaction rating of 10 (range 9-10), and no symptom of gastroesophageal reflux disease (GERD). The mean percentage excess weight loss (%EWL) was 77.1% (range 70.1-98.1) and the mean BMI was 25.7 kg/m2 (range 23-31). Conclusion: HM and RYGB are an excellent treatment for morbidly obese patients with achalasia. All patients reported a marked improvement of their dysphagia and no symptoms of GERD. The %EWL and BMI descent was very good and maintained through time, showing also an excellent control for morbid obesity.

Non-Bariatric Roux-en-Y Gastric Bypass.

Background: Roux-en-Y gastric bypass (RYGB) is frequently performed for weight loss purposes in the morbidly obese population. The popularity and acceptance of this procedure have increased the knowledge of the physiological (anatomical and functional) changes that this technique produces in the organism. RYGB improves gastric emptying and gastroesophageal reflux symptoms. Materials and Methods: We analyzed 6 patients in whom an RYGB was performed for non-bariatric purposes. Symptom questionnaire was used to evaluate response. Results: None of the patients qualified for bariatric surgery, as all had a body mass index (BMI) <35 kg/m2. Five patients were operated on for severe gastroesophageal reflux disease symptoms, and one for gastroparesis. All patients had good to excellent results, with marginal modification of their BMI. Conclusion: Non-bariatric RYGB can be considered in patients with functional diseases of the upper gastrointestinal tract, regardless of their BMI.

Resection of the Gastroesophageal Junction and Roux-en-Y Reconstruction as a New Alternative for the Treatment of Recurrent Achalasia: Outcomes in a Short Series of Patients.

Background: Heller myotomy and laparoscopic fundoplication represents the best treatment option for esophageal achalasia, with effective short- and long-term success. However, treatment options in patients in whom primary surgery failed represent a real challenge. We present the resection of the gastroesophageal junction (GEJ) along with a Roux-en-Y reconstruction as a treatment alternative. Materials and Methods: We analyzed the course of 5 patients with achalasia undergoing the resection of the GEJ along with a Roux-en-Y reconstruction for recurrent dysphagia after Heller myotomy and fundoplication, with at least 1 year of follow-up. Symptoms questionnaire and minuted esophagogram before and after treatment were performed in all the patients. Results: Five patients underwent resection of the GEJ along with a Roux-en-Y reconstruction. All the patients had dysphagia and 60% had regurgitations. Eighty percent of the patients had more than one previous redo surgery and 100% had had multiple dilations. Preoperative contrast esophagram of 3 patients show Stage II disease (mild and mark dilated esophagus) and 2 patients with Stage III disease (one esophageal curve and severe dilation). Manometry confirmed the diagnosis. At a mean follow-up of 34 months, all the patients reported a marked improvement in dysphagia, with median overall satisfaction rating of 9 (range 7-10), no symptom of gastroesophageal reflux disease (GERD), and good esophageal emptying in the postoperative contrast esophagram. Conclusions: The resection of the GEJ and Roux-en-Y reconstruction is an excellent treatment for recurrent dysphagia after Heller myotomy. All the patients reported a marked improvement of their dysphagia. No symptoms of GERD were documented after the surgery. This procedure should be taken into account as an alternative to esophagectomy for recurrent dysphagia.