Kulturer til nytte og beskyttelse

Transcription

1 24/09/2014 Kulturer til nytte og beskyttelse Protective cultures/beskyttelses kulturer kan idag bruges til give mervaerdi ved at saenke risiko for vaerdi nedsaettelse i rigtig mange af maelkens foraedlings step fra «jord til bord» 1

2 Definition of protective culture In the EU only active cultures that give extra consumer safety (= protects against pathogens) should be called protective as generally for fermented food the normal starter culture in the original way such products where developed are used (through it acid production ) to enhances shelf life, but naturally also may give change in flavour, texture, nutritional value etc. which has made them so popular Inactive cultures, sold only because of the contained metabolites to pro-long shelf life, are, legally seen, not cultures but are additive and principally must be labelled as such with E-number In other jurisdiction like as example USA fermented milk and fermented sugar, so principally just the metabolites or in-active cultures, can be added and labelled as such, hence possible to add high amounts of acids and bacteriocins for their anti-microbial effect 2

3 Raw milk «protection» Protective culture dairy applications* High ph fresh cheese «protection» against pathogens Cheese and fermented milk «protection» against Yeast & (foreign) moulds Cheese «protection» against Listeria m. Cheese «protection» against late blowing (Clostridia) Cheese «protection» against biogenic amines *examples/references from praxis can unfortunately not be given as the clients generally do 3not want us to tell about what they use, not even the smear culture clients in DK would give allowance!

4 Raw milk «protection» In countries with issues of hygiene, many small farms and too high storage temperature the milk quality is still not fantastic But also with good cooling, if the amount of psykrotroph bacteria becomes too high there are issues with the quality of the milk regarding flavour as well as stability in UHT because of high enzymatic activity still after heat treatment and/or because of extra heat resistant spore formers and maybe also with yield in cheese production Even if they all should be killed by pasteurization high counts of potential pathogen, Enterobact.s. and E.coli etc. are also not wanted Of this reason a protective strain for this use was selected, a strain that does not give negative flavour/texture impact in the final dairy products, does not inhibit the normally used starter cultures, but still has a strong inhibitory effect on the unwanted microorganism. Some examples show results from two of the factories testing it over a 5 day period in same silo milk split into two smaller tanks of which one was inoculated and the other not. 4

5 5 Raw milk «protection» ex.2, w.5x10e4/ml, f. 72h

6 6 Raw milk «protection» ex.2. w.5x10e4/ml,12-24h c.st.

7 7 Raw milk «protection» ex.2.w.5x10e4/ml,12-24h c.st.

8 8 Raw milk «protection» Ex.3. w. 5x10E4/ml, 6 C, 24h, total mesophilic count

9 9 Raw milk «protection» Ex.3, 5x10E4/ml, 6 C, 24h, psychrotrophe count

10 Raw milk «protection», discussion of results With adddition of 5x10E4 LRB/ml from concentrated culture, so no effect from carry over of metabolites, the total mesophilic count, psychrotrophic count and Enterobact./Coliform count all can be kept lower, though mostly only around one third for these counts, so not a huge difference in the raw milk before pasteurization, which was also not to be expected with a culture that does not grow much at 6+/-2 C, and especially not against G- bacteria that generally are not inhibited much by LAB apart from their acids. The difference though become very clear, when talking G+, pasteurization surviving bacteria, as the reduction after pasteurization, though same ph/ N in the milk, is app.1 log reduction! This lower count before and less survival of bacteria after pasteurization also has shown to give significant better stability of pasteurized and UHT milk as well as Queso blanco (not acidified soft cheese)! Mode of action not known fully, partly it seems to be from redox potential reduction, but else? No acids or significant amount of metabilites produced! 10

11 Fresh cheese «protection» Many fresh and soft not matured and weakly fermented cheese types with high ph (from above 6 and down to 5) have issues with out growth of unwanted microorganism, of which some can be pathogen (G- like Enterobact. incl. Salmonella and E.coli, Pseudomonas as well as G+ like Staph., Bacillus, Clostridia, Listeria etc. as well as yeast and moulds) if not produced clean enough or more correctly when produced very clean and then by default small unwanted contaminations happen, especially if not stored perfectly cold Of that reason different solutions have been tested and found effective for different types of cheeses, below example of a high ph fresh cheese made only with mild prt- ST as std., where different protective cultures where added +/- isolated G- contaminants often found. 11

12 Fresh cheese «protection», example S. STD = standard/reference product without any additions, ph 5,0-5,1 after 4 weeks, smell bad! Ditto at 8 C! On this production day no Pseudomonas was found, but usually yes. STD1 = with addition of 10E2 contaminants/g (isolations from problematic standard product), ph still 5,0-5,1 after 4 weeks at 14 C, smell very bad, ditto at 8 C! SA = addition of 10E6 protective culture SA/g, ph 5,0-5,1 after 4 weeks at 14 C, smell bad, ditto at 8 C! SA1 = addition of 10E2 contaminants/g + SA as above, ph 5,0-5,1 after 4 weeks smell very bad, ditto at 8 C! RH = addition of 10E6 protective culture RH/g, ph 4,4-4,5 after 4 weeks at 14 C, but characteristics incl. smell and flavour still very good, ditto at 8 C, though less ph drop! RH1 = addition of 10E2 contaminants/g +RH as above, ph 4,4-4,5 after 4 weeks at 14 C, but characteristics incl. smell and flavour still good, ditto at 8 C though less ph drop. CB = addition of 10E6 protective culture CB/g, ph 4,6-4,7 after 4 weeks, but characteristics incl. smell and flavour still good, ditto at 8 C, though less ph drop! CB1 = addition of 10E2 contaminants/g and CB, ph 4,6-4,7 after 4 weeks, but characteristics incl. smell and flavour still good, ditto at 8 C, though less ph drop. Mix = addition of 10E6 RH and 10E6 CB per g, ph 4,4-4,5 after 4 weeks at 14 C, but characteristics incl. smell and flavour still very good, ditto at 8 C, though less ph drop Mix1 = addition of 10E2 contaminants and RH+CB as above, ph 4,4-4,5 after 4 weeks at 14 C, but characteristics incl. smell and flavour still very good, ditto at 8 C, though less ph drop. 12

13 13 Fresh cheese «protection», T2 4 weeks at 8 C

14 14 Fresh cheese «protection», T2 4 weeks at 14 C

15 Fresh cheese «protection», discussion of results Lb.rhamnosus and/or Carnobact. ssp. and also strains of other Lb. species can give good inhibition of unwanted growth contaminants (naturally G+, but also G- bact. as well as yeast and moulds discussed later) Especially when the cold chain is broken the effect becomes higher as the protective cultures grow more, hence having exactly the wanted consumer protective effect and not only a shelf life prolonging effect (like from 4 to 6 weeks shelf life) Depending on strain (lactose +/-) the effect is more but then also gives stronger ph drop that depending on product may also influence the quality/flavour significantly when stored too warm Applied so far for different fresh cheese types (Sfatid, Cottage Cheese, soft Mozzarella a.o.) in Italy, Israel a.o. countries 15

16 LAB «protection» against yeast & moulds 16 LAB unfortunately has a limited effect against yeast & moulds, and the effect vary a lot from species to species and even strain to strain tested with over 20 typically occurring yeast like Kl. (2 Kl.m+ 2 x Kl.l), Sacch. (4 S.cer., 2 other), Cand. (2 C.kr., 2 other), 3 x Dh plus a few wild isolates not characterized, next to several mould species (mostly PR and GC), Many LAB strains tested and found having some activity, a few selected (and compared also to competitive products) and sold so far by Sacco, two Lb.rhamnosus and a Lb.plantarum that have reasonable good effect and not found to promote the growth of yeast strains, as some do as can be seen in the following example from the selection tests! Strangely enough for yeast the gas and yeasty flavour formation is more inhibited than the counts, hence a small inhibition and delay in growth can still give a good effect in prolongation of shelf life also for yeast and for the moulds especially, so sold well for white cheese and fermented milks Mode of action not really know!

17 17 LAB «protection» against yeast, example

18 18 LAB «protection» against moulds, example Geotrichum inhibition by LRH

19 LAB «protection» against moulds, Penicillum r. on yoghurt inhibited by different strains + Penicillium LC10 PBF02 LF07 LRH08 LA03 LRH02 LPC Penicillium inoculated 10 5 spore/ml Yeasts (blend of DH, KLM, SC)10 2 CFU/ml + Penicillium + yeasts After 10 days of incubation at 10 C + - The best Penicillium sporulation-inhibitor are: LRH08, LA03, LRH02, LPC17, LF07. Weak inhibition for PBF02, LC10

20 20 LAB «protection» against moulds, lab. example on process cheese

21 LAB «protection» against moulds, example cheese inoculated with LRH 21

22 Brine flora for surface «protection» againt moulds Not smeared Sterile brine Not smeared Yeast and Staph. in brine 7.0 surface ph (0-2mm) Mould flora of ripening room Smeared Sterile brine 5.0 ripening [days] sterile brining, complete starter 4b+1y sterile brining, no starter DH yeast + S. equorum in brines, starter 3b+0y DH yeast + S. equorum in brines, no starter

23 Smear strategies for Surface ripening old young old - young + SF starter SF starter + young - old

24 De-acidification of the cheese surface = Smear development 8,5 ph of cheese rind 8,5 ph of cheese rind 8,5 ph of cheese rind ph 7 ph [-] 8,0 7,5 7,0 6,5 6,0 5,5 5,0 ph [-] 8,0 7,5 7,0 6,5 6,0 6,0 5,5 5, time [days] 5, time [days] ph [-] 8,0 7,5 7,0 6,5 5, time [days] 7d 7d 7d whey separation after brining low medium high semi-hard cheeses: surface ph 7.0 = cell counts 10 9 cfu/cm 2

25 25 Wild cheese surface flora (/contaminants) lots of antibiotic resistance

26 Surface flora for red smear cheese Minimal composition for Tilsiter/Havarti a.o. (semi) hard type cheese Debaryomyces hansenii Staphylococcus equorum in brine! Brevibacterium linens Microbacterium gubbeenense in smear! Corynebacterium casei Minimal composition for soft cheese (Limburger) Geotrichum candidum in cheese milk! Debaryomyces hansenii Staphylococcus equorum in brine! Brevibacterium linens Microbacterium gubbeenense in smear! Minimal composition for für Harzer cheese (Gelbkäse) Kluyveromyces marxianus Candida krusei in the cheese milk! S. equorum in the Quark! Brevibacterium linens Corynebacterium sp. (?) in the smear!

27 Surface ripening cultures for protection Of above mentioned reasons today cultures are sold for inoculation of brine, giving significant effect against unwanted mould growth, ditto all the other single components, but also SRC blends are sold that give full red smear flora so that it is possible to inoculate young cheese giving perfect smear with only one days delay compared to the traditional back slopping Hence have no need of back slopping from old to young cheese, so giving much less risk of growth of unwanted pathogens or potentially such, nor of strains with antibiotic resistance, and less mould problems/ the possibility to stop cross contamination from old to young, if then used daily, as full flora or only as back up when needed is up to the cheese factory Used with very good results in AUT, CH, DE, DK, IT, SE 27

28 28 Bacteriocin producing protective cultures

29 29 Bacteriocin based «protection»

30 30 Bacteriocin producers against Listeria m.

31 31 Cheese «Protection» against Listeria m., example

32 32 Cheese «protection» against Listeria m., example

33 33 Cheese «Protection» against Listeria m., example

34 Bacteriocin producers against Clostridia etc. Generally bacteriocins are very easily degraded by proteolytic enzymes, incl. rennet in cheese, but it has been well documented by NIZO a.o. that nisin and similar class I bacteriocin can be produced in high enough amount and survive long enough to give a good inhibitory effect of Clostridia and other unwanted gas formers in cheese They though must be used in solutions giving high enough peptidase activity as the LAB resistant to these bacteriocin do not lyse in cheese and so do not give high enough de-bittering activity, or if prt+ even create bitter flavour in cheese! Hence the use needs that there are other LAB enclosed in the solutions that do release enough peptidase activity (Lb.helveticus/lactis or Lc.cremoris solutions can be used) As we talk single/defined strains of Lc.lactis/diacetylactis or St.thermophilus the solutions are rel. phage sensitive 34

35 35 Bacteriocin producers against Clostridia etc.

36 36 Bacteriocin producers against Clostridia etc.

37 Cheese «Protection» against Clostridia CO 2 H 2 Acetic acid Butyric acid Nitrates addition Lysozyme addition Cheese blowing Use of a strain producer of a bacteriocin active against Clostridia 15

38 Bac+ producers in a mini-cheese Industrial scale Gouda cheese production in laboratory scale Lab scale 1. Inoculum: 30 min -CaCl 2 -Starters -Clostridium tyrobutyricum spores -+/- bacteriocin producer strain/s 2. Renneting and curd cutting: 45 min 17

39 Bac+ producers in a mini-cheese Industrial scale Lab scale Stirring in vat 3. Stirring and whey off (30%): 30 min Low speed centrifugation 4. Stirring, heating and hot water addition: 40 min 5. Whey off: 60 min Pressing systems 6. Pressing: 30 min Low speed centrifugation 18

40 Bac+ producers in a mini-cheese Industrial scale Lab scale 7. Brining: 0 (to simulate cheese core) 8. Vacuum pack 10Kg vs. 15g 9. Ripening ~11-18 C ~20 C (to be sure!) 19

41 Sampling and cheese analysis: Bac+ producers in a mini-cheese 1 month ripening, no bacteriocin producer added -samples collected after 4 and 8 weeks of ripening; -free fatty acid extraction (FFA) and Gas Chromatography analysis for the detection and the quantification of the butyric acid; -well diffusion assay of small portion of cheese; 20

42 Bac+ producers in a mini-cheese, examples of results with some single bac+ strains together with DL-starter Mini-cheese added with single bacteriocin producer Lactococcus lactis strain: C) + DBC32 (subsp. lactis isolated from wastewater, Nisin Z producer) D) + SL28 (Nisin A) E) + SD66 (Nisin A) F) + SL147 (Lacticin 481 and Nisin Z) G) + U6_3 (lacticin 3147) H) + N245 (subsp. cremoris, isolated from grass, producer of unknown molecules) A) NOT added Bacteriocin producer (considered as 100%) 23

43 Bac+ producers in a mini-cheese, examples of results with some bac+ strains together with DL-starter Mini-cheese added of COUPLE of bacteriocin producers: I) + U6_3 and SL147 (Lacticin 3147 and Lacticin 481 producers) L) + SL28 and U6_3 (Nisin A and Lacticin 3147) M) + SL28 and SL147 (Nisin A and Lacticin 481) N) + U6_3 and SD66 (Lacticin 3147 and Nisin A) O) + SL28 and DBC32 (Nisin A and Nisin Z) A) NOT added Bacteriocin producer (considered as 100%) 24

44 Cheese «Protection» against Clostridia 44 The work done over the last years has given us a range of culture solutions that can be used to inhibit Clostridia and other nisin (as well as lactococcin and thermophilin) sensitive gas forming MO that may course spoilage in soft, semi-soft, semi-hard and hard cheeses, a by NIZO well documented solution, even for cheese made from milk of silage fed cows without nitrate/lysozyme Depending on cheese type, wanted flavour and technology this is not always the optimal solution or not always giving effect enough against Clostridia or de-carboxylating LAB (producing biogenic amines), of which reason we also selected as well as have licenced strains in of NSLAB from KU Science that also can inhibit Clostridia and other late blowing microorganism Last is mostly enough to give the needed extra effect when the milk is bactufuged/microfiltered, but mostly not in cheese made from milk from silage fed cows if no nitrate/lysozyme used as we have many examples from around the world showing!

45 45 NSLAB used for Cheese «Protection» against Clostridia

46 46 NSLAB used for Cheese «Protection» against late blowing

47 47 NSLAB used for Cheese «Protection» against late blowing

48 48 NSLAB used for Cheese «Protection» against late blowing

49 Collaborators The many new developments within protective cultures on which Sacco s range build have come from collaborations with many institutes around the world of which I will just mention a few that should have special thanks: Inst. Microbiologia, Unicat, Piacenza, IT Inst. f. Mikrobiologie u. Biotechn., MRI, Kiel, DE Inst. f. Microbiologie, ETH, Zuerich, CH Teagasc, Fermoy, IE Dept. o. Food Science, KU Science, Kbh.,DK 49 But also many others incl. colleges and our customers around the world that have helped us with the industrial testing

50 Thank you very much for listening! Clerici-Sacco Group Sacco S.r.l. Caglificio Clerici S.p.A. 50