Opinion on the Safety of BioProtein® by the Scientific Panel on Animal Feed of the Norwegian Scientific Committee for Food Safety

BioProtein® (BP) is a trademark for single cell (bacteria) protein, based on conversion of methane, with the addition of ammonia and oxygen, to a protein source. BP is produced by Norferm AS in Norway, and has been authorized by the EU as a protein source in animal feeds since 1995, for fattening pigs (8%), calves (8%) and salmon (19-33%). Significant immune effects were revealed in a toxicity study performed in rats fed a nucleic acid reduced BP product (NABP) and thereafter, similar, but less severe effects were also found after feeding with untreated BP. Additional studies confirmed increased mesenteric lymph node weights in cats and foxes. Due to the undesirable effects and also due to applications for extended use, BP has been assessed by the Scientific Committee on Animal Nutrition (SCAN) and EU’s Scientific Committee on Food (SCF) in 1995, by SCAN in 2001 and 2003 and by the European Food Safety Authority (EFSA) in 2005. The EU memberstates United Kingdom, France and Finland have also conducted assessments. The EU approval from 1995 remains unchanged. The Norwegian Food Safety Authority requested the Norwegian Scientific Committee for Food Safety (VKM) to assess the risk of using BP as a protein source in feedingstuffs, both for the animal categories already authorized and for extended use to pet animals, chickens and pigs from weaning to slaughter. The Norwegian Scientific Committee for Food Safety was asked to consider all existing documentation. Based on all documentation enclosed with the request from the Norwegian Food Safety Authority and published scientific articels, an opinion on the safety of BP assessed by the VKM panel on Animal Feed was published on 20 March 2006 (05/608-final-rev1). The Producer made a complaint regarding this opinion and claimed that not all documents on BP had been evaluated. The Norwegian Food Safety Authority then requested VKM to revise its opinion on the risk of using BP as a protein source in feedingstuffs, based on 17 documents previously not available to VKM, in addition to the 20 documents included in the opinion published on 20 March 2006. BP is composed of a protein with a somewhat different amino acid compostion compared with fish meal, but BP and fish meal have more similarities in amino acid content than soybean meal. BP has relatively high level of nucleic acids, phospholipids, lipopolysaccarides and minerals. Effect studies with BP have been conducted in rats/mice, pigs, chicken, cats, foxes, and salmon. Most of the concern regarding the side effects of BP in feed is related to the immune response. The main findings include changes in weight and morphology of mesenteric lymph nodes, followed by induction of specific antibodies. Histopathological examination after feeding with NABP also revealed changes in the intestines and several internal organs indicating systemic effects. The Producer claims that the immune response seen in BP-fed mice/rats is most likely a normal response to ingestion of large doses of a foreign antigen, and further, that oral tolerance towards this protein is induced over time. However, these interpretations are not adequately supported by the supplied documentation. A tendency towards adaption might be indicated in some of the studies, other results argue against tolerance induction. It is unclear whether the content of phospholipids, lipopolysaccarides, nucleic acids or the protein structure, or the combination of these compounds is responsible for the immunological changes observed. However, the particulate structure of BP has been shown to influence the observed immune response as the systemic immune response was avoided by ingestion of BP free of whole cells. The studies conducted in target species have not included adequate examinations of the immune effects from ingestion of BP. Concerning terrestrial species, no histopathological effects were revealed in the pig, chicken, cat or fox studies. However, increased mesenteric lymph nodes were reported in cats and foxes fed BP. In the remaining studies the main focus has been on production parameters; weight gain, feed intake, feed efficiency, metabolism of nutrients, observation of clinical health, and product quality. When the contents of amino acids were balanced, the inclusion of low levels of BP (9%) tended to stimulate growth in pigs and the same tendency was found in chicken with 6% BP. Higher feed levels of BP tended to cause a reduction in growth. In salmon, a dose dependent improvement of growth was reported in a short term experiment (8 weeks). However, in longer term experiments with salmon, depressed growth and increased liver weight were observed in freshwater at 19% BP with no-effect-level at 10%. In seawater studies, a tendency of reduced growth was found in salmon fed with 20% BP in the diet, and BP levels of 27% and higher levels resulted in significantly reduced body weight. Furthermore, levels of 27% BP and above in fish feed reduced specific immune responses, but increased lymphocyte response, and also tended to improve the survival after bacterial and viral infections. At 37% BP in the diet histopathological changes in the distal intestine, and reduced storage of glycogen and increased lipid deposition and liver weight were observed. No negative effects were seen in salmon in seawater at a feed level of 13.5% BP. The results indicate negative effects in salmon at BP levels in fish feed considerably lower than those currently approved (19 and 33%, in feed for salmon in fresh and sea water, respectively). To conclude, in terrestrial target species documented effects of BP include reduced weight gain and increased weight of mesenteric lymph nodes. In the more thoroughly studied species the rat, findings incluse histopathologic effects in mesenteric lymph nodes, changed humoral immune responses, increased weight of other lymphoid tissue (spleen), increased level of neutrophile granulocytes, and slight leakage of hepatic and renal tubuli enzymes. In terrestrial target species, 6% BP in the feed is considered to be the highest inclusion level not to cause such effects. The results from the rat studies show a similar no-effect-level. In salmon, reduced weight gain, liver storage effects, changed humoral and celluar immune responses and histopathological effects in the intestine are documented. A 10% level of BP in fish feed is the highest level tested without causing such effects. There are relatively few valid studies for the risk assessment of BP in target species, and the designs of the assessed studies are very variable. Thus, there are qualitative and quantitative uncertainties concerning the effects of BP in target species. The Panel on Animal Feed is of the opinion that an inclusion level of BP of 6% in the diets to terrestrial target animals and a 10% maximum inclusion level in salmon feed (both for fresh- and seawater stages) would reduce the risk of potentially adverse effects in the animals. The risk associated with the human consumption of products from animals fed on BP is considered negligible. However, the production of single cell protein for feed production represents a relatively new scientific approach which implies precautionary handling.

Prospective production of fructose and single cell protein from date palm waste

BACKGROUND: Fructose and single cell protein are important products for the food market. Abundant amounts of low-grade dates worldwide are annually wasted. In this study, highly concentrated fructose syrups and single cell protein were obtained through selective fermentation of date extracts by Saccharomyces cerevisiae. RESULTS: The effect of air flow (0.1, 0.5, 0.75, 1, 1.25 and 1.5 vvm) and pH (4.5, 4.8, 5, 5.3 and 5.6) was investigated. Higher air flow led to lower fructose yield. The optimum cell mass production of 10 g/L was achieved at air flow of 1.25 vvm with the fructose yield of 91%. Similar cell mass production was obtained in the range pH of 5.0­5.6, while less cell mass was obtained at pH less than 5. Controlling the pH at 4.5, 5.0 and 5.3 failed to improve the production of cell mass which were 5.6, 5.9 and 5.4 g/L respectively; however, better fructose yield was obtained. CONCLUSIONS: Extension of the modified Gompertz enabled excellent predictions of the cell mass, fructose production and fructose fraction. The proposed model was also successfully validated against data from literatures. Thus, the model will be useful for wide application of biological processes.

Influencing factors on single-cell protein production by submerged fermentation: a review

Since more than twenty years ago, some species of bacteria and fungi have been used to produce protein biomass or single-cell protein (SCP), with inexpensive feedstock and wastes being used as their sources of carbon and energy. The role of SCP as a safe food and feed is being highlighted more because of the worldwide protein scarcity. Even though SCP has been successfully commercialized in the UK for decades, study of optimal fermentation conditions, various potential substrates, and a broad range of microorganisms is still being pursued by many researchers. In this article, commonly used methods for the production of SCP and different fermentation systems are briefly reviewed, with submerged fermentation being highlighted as a more commonly used method. Emphasis is given to the effect of influencing factors on the biomass yield and productivity in an effort to provide a comprehensive review for researchers in related fields of interest.

Effect of initial pH of medium with potato wastewater and glycerol on protein, lipid and carotenoid biosynthesis by Rhodotorula glutinis

Background: Rhodotorula glutinis is capable of synthesizing numerous valuable metabolites with extensive potential industrial usage. This paper reports the effect of initial culture medium pH on growth and protein, lipid, and carotenoid biosynthesis by R. glutinis. Results: The highest biomass yield was obtained in media with pH 4.0­7.0, and the value after 72 h was 17.2­19.4 gd.w./L. An initial pH of the medium in the range of 4.0­7.0 has no significant effect on the protein (38.5­41.3 g/100 gd.w.), lipid (10.2­12.7 g/100 gd.w.), or carotenoid (191.7­202.9 µg/gd.w.) content in the biomass or on the profile of synthesized fatty acids and carotenoids. The whole pool of fatty acids was dominated by oleic (48.1­53.4%), linoleic (21.4­25.1%), and palmitic acids (13.0­15.8%). In these conditions, the yeast mainly synthesized torulene (43.5­47.7%) and ß-carotene (34.7­38.6%), whereas the contribution of torularhodin was only 12.1­16.8%. Cultivation in medium with initial pH 3.0 resulted in a reduction in growth (13.0 gd.w./L) and total carotenoid (115.8 µg/gd.w.), linoleic acid (11.5%), and torularhodin (4.5%) biosynthesis. Conclusion: The different values of initial pH of the culture medium with glycerol and deproteinized potato wastewater had a significant effect on the growth and protein, lipid, and carotenoid biosynthesis by R. glutinis.

Research advances in quantitative detection of single cell protein / 中国药科大学学报

Proteins presence and differences of the expression level can clarify the physiological or pathological changes in organisms;so the quantitative detection of proteins is vital for disease mechanism research;diagnosis and prognosis evaluation.Traditional protein quantitation methods at the tissue level reflected the average protein expression in cells;but ignore the differences between individual cells.In contrast;approaches for quantitative detection at single-cell level can better reflect the differences.Recently;a number of approaches for such detec-tion have been proposed;including microfluidics;microwell-based technology;optical fiber nanobiosensor;activity-based probe technology and mass spectrometry.The principles;advantages and drawbacks of these approaches are briefly introduced in this review.

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

Producción de biomasa de Saccharomyces cerevisiae y Candida utilis usando residuos de pulpa de Coffea arabica L

Los avances en biotecnología industrial ofrecen oportunidades potenciales para la utilización económica de residuos agro-industriales tales como la pulpa de café, material mucilaginoso, fibroso (producto secundario) obtenido durante el proceso húmedo o seco del beneficio de las cerezas de café. El propósito de este trabajo fue utilizar los residuos de la pulpa de café, rico en materia orgánica, como sustrato para la producción de biomasa de levaduras por procesos de fermentación aeróbica. Los residuos de café se sometieron a hidrólisis con una solución de ácido sulfúrico al 2%, en una relación 10:1 (líquido:sólido), con un tamaño de partícula ≤ 2 mm., operando a presión atmosférica, ebullición a reflujo, durante 4 horas. El extracto ácido se filtró y se ajustó a pH 4,5 y luego se esterilizó a 120 ºC por 15 minutos. La fermentación se realizó con Saccharomyces cerevisiae y Candida utilis, medio de producción extracto de café enriquecido con sales nutritivas. Se formularon diferentes medios de producción (1,2,3 y 4), siendo el N°3, enriquecido con extracto de café hidrolizado, 1L; urea, 3g/L; fosfato ácido de potasio, 2g/L; extracto de malta, 1,3g/L y melaza, 30g/L, el cual aportó los mejores resultados. El tiempo total de fermentación fue de 8 horas. Se obtuvo 10g/L de biomasa con un incremento proteico de 7,39 a 42,5%. Se puede concluir que la pulpa de café constituye un sustrato adecuado para obtener biomasa o proteína unicelular, que podría ser destinada como suplemento en formulaciones para alimentación animal.

The advances in industrial biotechnology offer potential opportunities for the economic use of agro-industrial remainders such as the coffee pulp, mucilagenous, fibrous material (secondary product) obtained during the humid or dry process of the benefit of the coffee cherries. The intention of this work was to use the remainders of the pulp of coffee, rich in organic matter, like substrate for the production of biomass of leavenings by processes of aerobic fermentation. The coffee remainders were put under hydrolysis with a sulfuric acid solution to 2%, in a 10:1 relation (liquid: solid), with a size of particle ≤ 2 mm, operating to atmospheric pressure, boiling to ebb tide, during 4 hours. The acid extract filtered and it adjusted to pH 4,5 and then it sterilize to 120 ºC by 15 minutes. The fermentation was made with Saccharomyces cerevisiae and Candida utilis, the production means: extract of coffee enriched with nutritious salts. Different means from production were formulated (1,2,3 and 4), being the N°3, enriched with extract of hydrolyzed coffee, 1L; urea, 3g/L; acid potassium phosphate, 2g/L; extract of Malta, 1,3g/L and molasses, 30g/L, which contributed the best results. The total time of fermentation was of 8 hours. 10g/L of biomass with a protein increase from 7.39 to 42.5% was obtained. It is possible to be concluded that the coffee pulp constitutes an suitable substrate to obtain biomass or unicellular protein, that could be destined like supplement in formulations for feeding animal.

Industrial derivative of tallow: a promising renewable substrate for microbial lipid, single-cell protein and lipase production by Yarrowia lipolytica

The aim of the present study was to assess the potential of valorisation of a solid industrial derivative of tallow, composed of saturated free-fatty acids ("stearin"), by fermentations carried out by the yeast Yarrowia lipolytica ACA-DC 50109 in order to produce microbial lipid, biomass and extra-cellular lipase. High quantities of biomass were produced (biomass yield of around 1.1 ± 0.1 g of total biomass produced per g of fat consumed) when the organism was grown in shake flasks, regardless of the concentration of extra-cellular nitrogen present. Cellular lipids accumulated in notable quantities regardless of the nitrogen availability of the medium, though this process was clearly favoured at high initial fat and low initial nitrogen concentrations. The maximum quantity of fat produced was 7.9 mg/ml corresponding to 52.0% (wt/wt) of lipid in the dry biomass. Lipase production was critically affected by the medium composition and its concentration clearly increased with increasing concentrations of fat and extra-cellular nitrogen concentration reaching a maximum level of 2.50 IU/ml. Lipase concentration decreased in the stationary growth phase. In bioreactor trials, in which higher agitation and aeration conditions were employed compared with the equivalent trial in the flasks, significantly higher quantities of biomass were produced (maximum concentration 30.5 mg/ml, yield of 1.6 g of total biomass produced per g of fat consumed) while remarkably lower quantities of cellular lipids and extra-cellular lipase were synthesised. Numerical models successfully simulated both conversion of substrate fat into biomass and production and subsequent hydrolysis of extra-cellular lipase and presented a satisfactory predictive ability verifying the potential for single-cell protein and lipase production by Yarrowia lipolytica ACA-DC 50109. In all cultures, the mycelial form of the culture was dominant with few single cells present.

STUDIES ON THE OPTIMAL MIXED PROPORTION OF INOCULATION IN PRODUCING SINGLE SELL PROTEIN / 微生物学通报

Using apple pomace to produce single cell protein (SPC) with three and four strains respectively, the optimal mixed proportion of inoculation, one of the main factors of multiple strains' fermentation to produce SPC, was studied The experiment and analysis indicated that a practicable method to get the optim al mixed proportion of inoculation was obtained, which will perfect correlative researches of this field