Z-Enology  Z-Wine Assay TM                                            Item #s:  Z-Brett 24

Protocol for Brettanomyces                                                                                            Z-Brett Basic 24

Distributed by Unitech Scientific LLC                                                                                             Z-Brett Basic 150

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Introduction

This Z-Wine Assay is an immunoassay (antibody-based) system for rapid detection of Brettanomyces and other species of spoilage yeast.  Z-Brett is intended to monitor the growth of Brettanomyces in settled wines following initial racking 1, 2; a monthly barrel monitoring program can detect Brett growth prior to sensory Brett detection.  Generally this assay is very robust, and can be performed in any environment (“field test”).  Z-Brett is a screening test; a confirmatory test is recommended prior to initiating expensive remediation 3.

Kit includes

·          Multiple 6-well Z-Grip™ Chips: 4 chips - for 48 wine results (24-duplicates: Z-Brett 24); 25 chips for 300 results(Z-Brett 150)

·          Decolorizer (A)  

·          Suspension Buffer (B)

·          Destain / Blocker (C)

·          Anti-Brett (D)

·          Buffer (E)

·          Conjugate (F)

·          Developer Diluent (G1)

·          Developer Active agent (G2)

·          Supplies (Z-Brett 24 kit only)

o         4 x Petri Dishes

o         25 low-retention Pipette tips

o         3 15mL measuring tubes

o         1 Sharpie pen

o         Disposable Pipettes, 1mL & 3 mL

Accessories - not supplied with Kit   (available from Unitech)

·          Centrifuge e.g. Microfuge Unitech PN: LX-100, $180) 1.5mL

                   centrifuge capability required

·          Micro-pipette 5 µL adjustable (available from Unitech) required

·          UniBrett Sampler (5, 10, or 15-foot - Tank/Barrel sample collection) Starting at $45  - bottom sampling greatly increases Brett recovery

Sampling, Sample Preparation
Collect two independent samples from within an inch of the bottom of tank or barrel 4, sample size 1 mL (to 100mL - for ultra senstive sample prep, refer to Note 5) and pipette into 1.5mL microfuge (or appropriate centrifuge) tube.

Sample Prep (Photos)   

a)       Centrifuge (1.5 ml microfuge tube for 5-minutes , larger samples for 15’ or more depending on centrifuge speed. Balance tubes to avoid damaging the rotor.

b)       Remove supernatant using the fine-tip 1mLdisposable pipette provided (or Pasteur pipette / aspiration flask / vacuum pump.)  

  • Avoid disturbing pellet at the bottom of the tube.  If pellet is not visible, allow about 50uL of liquid to remain undisturbed.

c)        Add 1ml Decolorizer (A) (Photos) and suspend cells by vortex mixing or pipette/re-pipette 5-10 times.

d)       Repeat one time the wash steps a) , b) & c) above. 

e)       Suspend pellet in 1 drop (40 uL) of Suspension Buffer (B).  Mix to resuspend pellet. (for ultra senstive ‘heat-conditioning’, refer to Note 6.)

Procedure

STEP 1 – Apply Samples & Dry  (Photos) 

a)       Record Sample I.D. on the chip to identify wine locations

b)       Mix samples well immediately before spotting.

o         It is VERY important to mix each sample immediately before application, since Brett settles quickly

c)       Place chip in petri dish; apply 5 µL to each well on the chip using a micro-pipette; do not touch chip with the tip

d)       Gently place entire chip in covered petri dish to dry overnight (be careful moving chips after spotting to prevent spots from running or smearing)

Optional Quick Drying Method – Place petri dishes in 37 -45˚C under incandescent light incubator or for rapid drying for at least 30 minutes.  

e)       Inspect chips for dryness.  When dry (dry wine spots appear glossy) proceed to step 2.

STEP 2 – Destain & Block   (Photos) 

For the following processing steps, the chip must be covered in liquid (i.e.10 mL). Place dried chip in Petri dish provided.

a)       Using measuring tube provided, pour 10 mL Destain/Blocker (C) carefully over chip, so that the chip is covered in liquid.

b)       Agitate the dish rapidly from side-to-side until wine color diffuses away (approximately 1-2 minutes).  Carefully observe wells for air bubbles on the membrane.  If necessary squirt  Destain/Blocker (C) with disposable pipette to remove air; do not touch membrane with pipette tip.

c)       Allow the chip to rest in Destain/Blocker for about 15 minutes, occasionally shake gently to mix; avoid bubbles.

d)       Repeatedly squirt Destain/ Blocker (C), using disposable

pipette provided, directly onto any persistant stain(s) until only very light (or no) color is present.  Note that light stains will fade after processing.

STEP 3 – Anti-Brett Antibody  (Photos)        Sequentially pipette 240 uL  (or 6 drops) of Anti-Brett (D) and 240 uL  (or 6 drops) of Conjugate (F) into the petri dish (beside the chip) containing Destain/Block. Mix  periodically (2 or 3 times) over 30 min.

STEP 4 - Conjugate   (Photos)        Discard all liquid from petri dish. Using a clean (or rinsed) measuring tube, pour 10 mL Buffer (E)  into dish to cover chip.  Pipette 240 uL  (or 6 drops)  Conjugate (F) into the petri dish and mix by hand periodically (2 or 3 times) over 30 min.

STEP 5 – Wash   Discard all liquid from petri dish.  Using a clean (or rinsed) measuring tube, pour 10 mL of Buffer (E) into dish to cover chip.  Mix gently about 1 minute, discard liquid. Repeat wash two times for a total of 3 washes; discard liquid.

STEP 6 – Develop Color   (Photos)       NOTE:  Active Developer G-2 is hazardous. Handle, & dispose of pipette tips, with care!  Prepare 10mL of Developer Mixture by combining 10 mL of Developer Diluent (G-1) and 0.1 mL Active Developer (G-2) in a clean measuring tube (or, simply pour G-2[0.6 ml] into G-1 [60mL];use within 3 weeks.)  Pour 10 mL Developer Mixture into dish to cover chip; wait 20 minutes.  Discard liquid.

STEP 7 –Rinse Chip with cold tap water for a few seconds; place upright at an angle & air dry (or use compressed air.)  (Photo) 

Interpret Results 

Procedural Controls & Test Validity  - The Z-Brett test is valid if both procedural controls are appropriate:

‘(-)’ is less intense than the 104 standard 7. and ‘(+)’ is dark, similar to the 105 & 106 Standards.

A sample is NEGATIVE or Equivocal if the average shade of the duplicate results is darker than the ‘(-)’ procedural control and lighter than the 104 Standard; this wine may be concentrated and retested using the Ultra-Sensitive Sample Prep.

A sample is POSITIVE if the average shade of the duplicate results is as dark or darker than the 104 internal standard.

Internal Standards at 104, 105 & 106 CFU/mL (pre-spotted) are for estimating sample concentration 7.

EXAMPLE Interpretation:   Assign CFU/mL values to your Samples equal to that of the Internal Standard of similar intensity. In the example shown, wine samples #2, 4 and 6 are negative, sample 5 has approximately 104 CFU/mLof Brett, sample #1 has 105 CFU/mLof Brett, and sample #3 has 106 CFU/mL of Brett.

Interpret Alternate Ultra Sensitive Prep
Estimate the CFU/mL of each sample by first identifying the Internal Standards of similar intensity.

For 10 mL of wine: Assign CFU/mL values to your Samples equal to 10% of the Internal Standard of similar intensity (i.e. divide that standard by 10 since, in this ultra-sensitive sample prep assay, samples are concentrated 200-fold instead of 20-fold for the pre-spotted Internal Standards.)   Any signal as dark as the 104standard is considered positive.

For 100 mL of wine:  Assign CFU/mL values to your Samples equal to 1% standard (i.e. divide by 100 since samples are concentrated 2000-fold instead of 20-fold for the Internal Standards.)   Any signal darker than 104standard is considered positive.  Background is slightly increased with 100 mL of wine. 

EXAMPLE InterpretatioN::  Refer to the example photo – if 100 mL of wine were processed,  #2, 4, 5 and 6 are negative, sample #1 has  103  CFU/mL of Brett (i.e. 105/102 =103), and sample #3 has 104 CFU/mL of Brett  (i.e. 106/102 =104.)

Interpret Heat Conditioning Ultra Sensitive Prep

(refer to footnote 6). Signal is enhanced by conditioning cell walls with heat and interpretation is often clearer.  Use interpretation guidelines above.

Antibody Specificity & Cross Reactivity
Brettanomyces Strains:  The Z-Brett test system detects all common strains of Brettanomyces/Dekkera yeast.

Other Spoilage Yeast Species:  Z-Brett also gives a positive response to a few other non-Saccharomyces yeast known to produce spoilage of wine and beer 8, 9.  

Normal Flora:  The Z-Brett system does not react with Saccharomyces cerevisiae 10, responsible for primary fermentation nor does the Z-Brett system react with commonly used microbes responsible for mali-lactic (secondary) fermentation 11 at concentrations typical of racked wine. 

 

 

 

Developed by:  Z-Enology Anaheim, CA.                             

 Distributed by: Unitech Scientific LLC
                            Hawaiian Gardens, CA  p. 562 924-5150       

              http://unitechscientific.com 

              sales@unitechscientific.com

 

Z-Brett_instruction_US_5-12-09

[1] Fermentation lees and actively fermenting must is not a suitable sample for Z-Brett testing. The high solids load in these samples decrease the binding of Brett to the Z-Brett membrane.  Additionally, the extreme abundance of Saccharomyces in these samples also interferes with Z-Brett detection. 

[2] The Z-Brett antibody does not detect fermentation yeast  (e.g. Saccharomyces) at levels found in most racked wines which have completed fermentation.  Furthermore, Brettanomyces is typically not found in wine prior to this vinification stage; it is an extremophile and does not thrive in the presence of Saccharomyces. 

[3] The Z-Brett system detects recently killed Brettanomyces.

[4] Brettanomyces/Dekkera tend to settle quickly in wine.  When present, Brett is most abundant within 1 inch of the bottom of the wine barrel or tank, and in sediment. Wine samples (exclusive of fermentation lees) collected from this location are ideal for Z-Brett detection. 

[5] Alternate Ultra Sensitive Sample Prep  

a)  Collect 10-100 ml sample. Concentrate cells by centrifugation.

b)  Suspend in 1 ml wash buffer. Transfer to 1.5 ml tube

c)  Proceed as described in Sample Prep Method #1

 

 

 

This procedure delivers 10-times (or 100-times) the cells to the Z-Brett chip; the CFU value of Brett will be 10-fold (or 100-fold) that of the corresponding Internal Standard.   Refer to the ‘Interpretation – Alternate’ Section.

[6] Alternate Ultra Sensitive ‘Heat Conditioning‘

Heat the suspended pellet (Sample Prep Step C) in boiling water 5-minutes, cool to room temperature, & spot  as above.

 

[7]  The (-) Control, pre-spotted with Saccharomyces at 107 CFU/mL, is minimally reactive with detection reagents.  Saccharomyces cerevisiae is the only yeast found at significant levels in healthy wine.  The high sensitivity and defined antibody specificity of the Z-Brett test alerts the winemaker to the presence of these yeast spoilage organisms, so that the appropriate treatments may be undertaken in a timely manner.  

 

9 e.g. Issatchenkia Orientalis, Zygosocchi Bailii, Pichia Sp., Candida Glabrata, Issatchenkia Orientalis, Torulaspore Pretoriensis

[10] Saccharomyces strains tested include ATCC, BM-45, DV-100, 12323, UVAFERM-43, Premier Cuvee, T73. 

[11] M-L bacteria tested include Lactobacillus plantarum,  Oenococcus oenos Nia,  Pediococcus sp. (damnosus),  Lactobacillus hilgardii,  Oenococcus oenos.