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WO2019048997A1 - Procédé pour la production d'une biomasse microbienne et le traitement du sérum du lait ou d'autres résidus de l'industrie laitière - Google Patents

Procédé pour la production d'une biomasse microbienne et le traitement du sérum du lait ou d'autres résidus de l'industrie laitière Download PDF

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Publication number
WO2019048997A1
WO2019048997A1 PCT/IB2018/056630 IB2018056630W WO2019048997A1 WO 2019048997 A1 WO2019048997 A1 WO 2019048997A1 IB 2018056630 W IB2018056630 W IB 2018056630W WO 2019048997 A1 WO2019048997 A1 WO 2019048997A1
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Prior art keywords
process according
ultrafiltration
feed
fermentation
batch
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Ceased
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PCT/IB2018/056630
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English (en)
Inventor
Rosita PAVONE
Erika ANDRIOLA
Maria PISANO
Isabella PISANO
Maurizio Bettiga
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Bioinnotech Srl
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Bioinnotech Srl
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to a process for the production of microbial biomass, in particular yeasts and milk bacteria, starting from whey and other waste of dairy industry.
  • the process according to the present invention represents a new approach for managing dairy waste limiting waste, reducing disposal costs by guaranteeing a low environmental impact.
  • it provides an alternative method for the production of microbial biomass.
  • yeast production is performed by using molasses, sugar residues from processing of beet and sugar cane, with a supplement of nitrogen source.
  • the standard process is a fermentation in fed-batch mode, during which molasses is added to the bioreactor gradually soon after the addition of cells.
  • the profile therewith the feed of molasses is provided in the industrial processes is not wholly known.
  • Several attempts are currently performed with the purpose of further improving this productive process by inserting new raw materials as fermentation substrates or variations in the process type (Patent EP0821057B1 , Baker's yeast production from molasses/cheese whey mixtures; Ferrari et al. 2001 ).
  • Patent CA1 133753A The acid whey, Patent CA1 133753A which is pre-treated thermally (Patent: US07890513; Improved Saccharomyces Cerevisiae Growth on Cheese Whey by Controlling Enzymatic Lactose Hydrolysis.; Pisano et al. 2015) or by ultrafiltration (UF) to remove the protein fraction (Patent CA1 133753A).
  • the membrane treatment process today is widely used for concentrating serum proteins, sugars or the serum itself, however nowadays these concentrated fractions are usually dried up and put into the market as such.
  • the patent CA1 133753A of 1978 describes a process wherein, after the batch fermentation step, a continuous step is performed during which serum and yeast are inserted constantly (constant time and volumes; volumes of feed equal to the volumes of product which is removed), for a total duration of about 18 hours of batch process and 4 hours of continuous process.
  • the process for the production of yeasts is a typically aerobic process, performed at a temperature of about 30°C under pH control conditions.
  • Many fermentation processes provide a pH continuous control to avoid a stress on the growing cells (Champagne et al.1989; Patent CA1 133753A).
  • this control requires considerable cost and energy consumption in an industrial optics.
  • the fermentative process is preceded by a step of pre-cultivation of cells which can be performed in synthetic growth medium or directly in serum.
  • the article of Ferrari et al. Biotechnology letters vol. 16 pages 205-210 describes a process for the production of bioethanol wherein the whey is firstly subjected to an atomisation process by spray-drying to obtain lyophilised lactose which is added to a second amount of whey as such in order to obtain a higher concentration of lactose in serum.
  • the patent application WO20141 164 describes a mode for recovering lactose starting from a liquid source containing lactose through crystallization and optional filtration steps for the removal of cells, proteins, polypeptides, polysaccharides, lipids, ions and salts.
  • the technical problem placed and solved by the present invention is then to provide a process for preparing yeasts and lactic bacteria by using as growth medium the whey or other milk waste of dairy industry allowing to obviate the drawbacks mentioned above and allowing at the same time to reduce the polluting organic load of the whey or of other milk waste of dairy industry.
  • the present invention then relates to a process for the treatment and re-qualification of dairy waste aimed at producing microbial biomass. Such problem is solved by a process according to claim 1 .
  • the whole milk waste of dairy industry is subjected to a pre-treatment (microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), optionally reverse osmosis (Ol)) and afterwards to a fermentation step.
  • the first three steps make homogeneous the raw material which is then used for fermentation.
  • MF allows to remove coarse particulate and fats
  • UF removes the protein fraction which is then lyophilised and used as food supplement
  • NF allows to concentrate lactose by removing part of salts; this fraction together with the liquid fraction of UF is then used in the fermentation step.
  • the step of reverse osmosis allows to produce demineralized water which is inserted as process water in the steps for keeping the fermentative process or washing temperature.
  • the fermentation process provides two steps, a first batch step performed with the deproteinized serum deriving from UF and a second fed-batch step wherein a feed controlled in serum (or other waste) deriving from UF or NF is provided.
  • the used raw material both in batch and fed-batch, is hydrolysed enzymatically, by adding beta-galactosidase, contemporary to the fermentative process: in this case one speaks about SSF (simultaneous saccharification and fermentation) process.
  • This combination of pre-treatment and fermentative process has resulted to be very effective for the production of microbial biomass of different type, in particular yeasts for different applications (Saccharomyces sp.; Kluyveromyces sp.; etc.) and lactic bacteria (Lactobacillus sp.).
  • the process according to the present invention then provides a valid alternative to the classical expensive disposal, having a high environmental impact, of dairy waste. At the same time, it provides an alternative method for the production of microbial biomass.
  • the pre-treatment and developed fermentative process together allow to reduce the presence of proteins, sugars and other substance constituting the polluting organic load of the whey, by allowing on one side to obtain market products of interest, on the other side to solve the problem of disposing this waste.
  • FIG. 3 summarizes the main parameters used in a preferred embodiment of the fermentation step of the process according to the present invention
  • Figures 4A and 4B show the course of the feed volumes with respect to the biomass growth (expressed in Optical Density, OD) during the fed-batch step.
  • Figure 2A describes the course of a feed with a UF Permeate
  • Figure 2B with a NF Concentrate.
  • the present invention relates to a process for the production of microbial biomass by means of whey and/or other milk waste of dairy industry.
  • the whey deriving from the dairy processing procedures, is a substrate rich in organic matter which then can be used in the fermentation processes wherein microorganisms use the existing carbon sources, in particular sugars.
  • One of the advantages of the process according to the present invention is the possibility of using both whey and other dairy waste, such as ricotta whey and buttermilk.
  • whey in particular one refers to the liquid fraction deriving from the formation of curd during cheese making; under the term ricotta whey one refers to the liquid fraction which is obtained after coagulation of ricotta, whereas under the term buttermilk the remaining waste water is designated deriving from the stretching process and from washing the stretched curd.
  • the process provides a step i) of microfiltration (MF) of whey and/or of other milk waste of dairy industry.
  • MF microfiltration
  • MF is performed by means of membranes with pores having diameter ranging from 0.1 to 1 pm capable of keeping the coarser particulate and the fats which would invalidate the subsequent growth step and the protein composition.
  • membranes made of polyethylene sulfone (PES) could be used.
  • the process provides a step ii) of ultrafiltration (UF) of the permeate obtained from MF.
  • the membranes of UF have a porosity ranging from 5 to 50 KDa, preferably 30 KDa, which do not allow the passage of the protein fraction ("UF concentrate”), but they let sugars, mineral salts and water (“UF permeate”) to go through.
  • the protein fraction concentrates and it can be recovered for lyophilization, drying or hydrolysis of serum proteins.
  • the US permeate is partially sent to fermenter.
  • the UF permeate is nanofiltrated (NF), for example with membranes ranging from 150 to 300 Da allowing to keep the lactose by concentrating it (“NF concentrate”), whereas it lets a portion of salts and water (“NF permeate”) to go through.
  • NF nanofiltrated
  • NF permeate can be subjected advantageously to reverse osmosis (01) or it can be used as such as process water for example in keeping fermentation temperature.
  • Ol process for example could be implemented by means of spiral-wound membranes made of PES with a flow mainly depending upon the used pumping system and its rate of flow.
  • the process according to the present invention provides a step iv) for fermenting a culture of microorganisms.
  • a step iv) for fermenting a culture of microorganisms Such step is herein described in details.
  • the fermentation step is performed under aerobic conditions with controlled flow of sterile air, for example between 0.5 and 5 L/min, at a temperature between 25 and 40°C and a stirring kept constant between 600 and 1000 rpm.
  • the fermentation could, or could not, provide the pH control, but it was observed that this does not induce significant variations on the yields of the produced biomass.
  • the cells are pre-cultivated preferably in synthetic growth medium, then transferred in a small volume of UF permeate and subsequently transferred in fermenter.
  • UF permeate and NF concentrate which are used as growth medium could have not to be sterilized thermally if the used membranes comply with current sterilization and sanitization criteria; otherwise they are subjected to suitable thermal sterilization.
  • a nitrogen source to the substrate used for the fermentative process; it is possible to add anyone of the nitrogen sources commonly used in the process for producing yeast (for example yeast extract, bactopeptone, ammonium sulphate, urea etc.).
  • the nitrogen concentration is selected so as not to be limiting but it is kept at a minimum level which does not compromise the process cost-effectiveness.
  • nitrogen sources in particular urea, bactopeptone, yeast extract and ammonium inorganic sources, were tested by evaluating the effect on the growth of microorganisms. These sources were tested in variable percentages, from 0.5 to 40% w/v. The highest concentrations guarantee higher biomass yields, but with greater costs. In fact, the minimum percentages, comprised between 0.05 and 5% w/v, were detected which guarantee a good microbial growth without invalidating the process yields significantly, but allowing considerable cost savings due to the reduction in the concentration of added nitrogen source. According to a preferred embodiment in fermentation a nitrogen source in a concentration between 0.05 and 5% w/v then will be used.
  • beta-galactosidase is added during fermentation, in particular at the beginning of the batch step in concentration proportional to the sugars existing in the culture medium.
  • lactose for example S. cerevisiae
  • beta-galactosidase is added during fermentation, in particular at the beginning of the batch step in concentration proportional to the sugars existing in the culture medium.
  • SSF processes the enzymatic hydrolysis is performed directly in the reactor wherein fermentation and growth of microorganism take place at the same time.
  • the enzyme is added at the beginning of the process, without impacting on process and plant cost-effectiveness, contemporary to the inoculum of cells.
  • the used enzyme amount is selected so as to avoid the accumulation of product (glucose and galactose) inhibiting the growth.
  • Variable amounts of enzyme were tested, from 5 to 5000 enzymatic units per litre (UE/L) to detect a range allowing the complete hydrolysis without inhibiting the process and with the minimum economic impact on the process.
  • the most suitable amounts resulted to be comprised between 800 and 2500 UE/L.
  • An additional new aspect of the process consists in the type of fed-batch which was developed: firstly, in the batch step, the cells are inoculated in a fixed volume of UF permeate, this step lasts from 2 to 8 hours. At the end of this step a feed step starts which can be performed with UF permeate or NF concentrate.
  • NF concentrate in a fermentative process is an important new aspect in this context. In fact, even the use of UF permeate allows the microbial growth, but, by performing the feecf with NF concentrate the working volumes are reduced considerably with a significant increase in the yields.
  • an addition of the beta-galactosidase enzyme in the feed volume is performed.
  • the flow of feed is increased gradually to provide an amount of sugars instantaneously proportional to the number of cells existing in the reactor.
  • This step can have a duration of 10-20 hours and if performed in this way, it allows the complete consumption of the sugars existing in the medium.
  • the variability in feed time depends upon the composition of the input waste; this allows a certain flexibility in using the substrates with different amounts of sugars, as it allows to customize the fermentation profile (time and feed volumes) based upon the substrate features.
  • the rate and amount of feed which are input in the reactor are calculated based upon: amounts of sugars in the feed, amounts of cells existing in the reactor when the feed starts and theoretical maximum yield (defined in advance and known).
  • amounts of sugars in the feed amounts of cells existing in the reactor when the feed starts and theoretical maximum yield (defined in advance and known).
  • the feed volume is defined according to the following equation: ⁇ * X * V 0 * ⁇ ( ⁇ * t)
  • C concentration of sugars in the growth medium expressed in glucose equivalents (g/L).
  • the microbial biomass is recovered by centrifugation or filtration.
  • the obtained microbial biomass is separated by centrifuge and it can be lyophilized or dried to obtain a powder product.
  • Example EXAMPLE 1 20 litres of whey, provided by a local dairy company, were subjected to an initial filtration process by means of a small-scale membrane system having a maximum yield of 21 L/min; with spiral-wound membranes made of PES. The first passage of MF, performed with membranes having a porosity of 1 pm, allows to remove the coarse particulate. During this passage a significant volume loss is not observed. MF permeate is subjected to UF with 30-KDa membranes.
  • MF permeate is made to circulate several times inside the membrane by allowing the formation of UF concentrate rich in the protein fraction (about a tenth of the initial volume) and deproteinized UF permeate which is then partially subjected to NF.
  • NF passage performed with 300-Da membranes allows to concentrate the sugar fraction; even in this case UF permeate is made to circulate inside the filtration system until obtaining a NF concentration equal to one fourth of the initial volume (5L).
  • the concentrations of sugars lactose, glucose and galactose
  • HPLC concentrations of sugars
  • b-galactosidase enzyme and the nitrogen source are added.
  • An inoculum of cells of S. cerevisiae is inserted in the fermenter so as to obtain a final optical density (OD) comprised between 0.5 and 5.
  • OD optical density
  • Such cells are pre- cultivated previously for about ten hours in 50 ml of synthetic growth medium and then transferred into 100 ml of sterile UF permeate added with b-galactosidase for further ten hours.
  • the system is subjected to continuous and controlled stirring of 800 rpm and the temperature is kept between 28 and 32° C; the air flow is kept at 1 .5 L/min.
  • the growth is monitored by measuring OD every two hours.
  • the batch step lasts 10 hours; at the end of this step the fed-batch step is started during which a feed of UF permeate is added for about 7 hours.
  • This feed is increased proportionally to the growth of the biomass in the reactor; in particular the feed volume which is added in a certain time unit is proportional to the amount of cells existing in the reactor; this guarantees a supply of sugars suitable to favour the growth of cells with the maximum performances by following an exponential course.
  • the fermentation proceeds for about further two hours by favouring the consumption of all sugars existing in the reactor.
  • the cell growth is monitored by measuring OD and the course of sugars in the medium is monitored by means of HPLC analysis.
  • the medium is collected and centrifuged for separating and recovering the microbial biomass.
  • the illustrated processes were tested by evaluating the effect on the growth of microorganisms of several nitrogen sources, in particular urea, bactopeptone, yeast extract and ammonium inorganic sources. These sources were tested in percentages variable from 0.05% to 40% w/v. The highest concentrations guarantee higher biomass yields but with higher costs. In fact, the minimum percentages were detected, comprised between 0.05 and 5%, which guarantee a good microbial growth without invalidating the process yields significantly, but allowing a considerable economic saving thanks to the decrease in the concentration of added nitrogen source.
  • suitable sources there are yeast extract and urea; the latter preferably is economically more advantageous, the performances being equal.
  • the illustrated processes were tested even by evaluating the effect on the growth of microorganisms of different concentrations of enzyme to detect the suitable concentrations which could not inhibit the growth by allowing even an economical advantage. Concentrations comprised between 50 and 5000 enzymatic units (UE) per litre were evaluated.
  • the present invention also relates to the microbial biomasses which can be obtained with the process according to anyone of the described embodiments. In particular it relates to biomasses of yeasts, for example of Saccharomyces in lyophilized or dried form.

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Abstract

La présente invention concerne un procédé pour la production d'une biomasse microbienne, en particulier de levures et de bactéries du lait, à partir de lactosérum et d'autres résidus de l'industrie laitière. Le procédé selon la présente invention représente une alternative valable à la mise au rebut coûteuse classique, ayant un impact environnemental élevé, de résidus de laiterie. En même temps, le procédé selon l'invention fournit une autre méthode pour la production d'une biomasse microbienne.
PCT/IB2018/056630 2017-09-06 2018-08-30 Procédé pour la production d'une biomasse microbienne et le traitement du sérum du lait ou d'autres résidus de l'industrie laitière Ceased WO2019048997A1 (fr)

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IT102017000099855 2017-09-06
IT102017000099855A IT201700099855A1 (it) 2017-09-06 2017-09-06 Procedimento per la produzione di biomasse microbiche ed il trattamento di siero da latte o altri reflui dell’industria lattiero casearia

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WO2019048997A1 true WO2019048997A1 (fr) 2019-03-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT201900021027A1 (it) * 2019-11-13 2021-05-13 Itest Srl Procedimento di produzione di formaggio a pasta filata

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011140649A1 (fr) * 2010-05-14 2011-11-17 Corporation Het - Horizon Environnement Technologies Procédé de fermentation d'un substrat en culture mixte destiné à produire une biomasse comestible par les animaux et/ou se prêtant à la consommation par l'homme
WO2014141164A1 (fr) * 2013-03-15 2014-09-18 Paul Stephenson Récupération de lactose

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011140649A1 (fr) * 2010-05-14 2011-11-17 Corporation Het - Horizon Environnement Technologies Procédé de fermentation d'un substrat en culture mixte destiné à produire une biomasse comestible par les animaux et/ou se prêtant à la consommation par l'homme
WO2014141164A1 (fr) * 2013-03-15 2014-09-18 Paul Stephenson Récupération de lactose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ISABEL ESPINOSA-GONZALEZ ET AL: "Heterotrophic growth and lipid accumulation of Chlorella protothecoides in whey permeate, a dairy by-product stream, for biofuel production", BIORESOURCE TECHNOLOGY, vol. 155, 1 March 2014 (2014-03-01), AMSTERDAM, NL, pages 170 - 176, XP055461899, ISSN: 0960-8524, DOI: 10.1016/j.biortech.2013.12.028 *
MARIO DANIEL FERRARI ET AL: "ETHANOL PRODUCTION FROM CONCENTRATED WHEY PERMEATE USING A FED-BATCH CULTURE OF Kluyveromyces fragilis", BIOTECHNOLOGY LETTERS VOLUME, vol. 16, 1 February 1994 (1994-02-01), pages 205 - 210, XP055461929 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT201900021027A1 (it) * 2019-11-13 2021-05-13 Itest Srl Procedimento di produzione di formaggio a pasta filata

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